UNIT 1: GENETICS: NUCLEIC ACID DNA

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23 Οκτ 2013 (πριν από 4 χρόνια και 17 μέρες)

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UNIT 1:
GENETICS:
NUCLEIC ACID

DNA

(Campbell & Reece:

Chapters, 5, 16)


LOCATION OF DNA IN
A CELL


Chromatin is a complex of
DNA and
protein
, and is found in the
nucleus

of
eukaryotic cells.


Histones

are proteins that are
responsible for the first level of DNA
packing

in chromatin


The
chromatin network
in the nucleus of
a cell will coil up tightly during cell
division and form individual
chromosomes
.


Chromosomes are always
duplicated

during this process (2 sets of identical
genetic information to ensure each cell
receives identical genetic info to the
parent cell during cell division).


A
duplicated chromosome
consists of 2
chromatids

attached to each other by a
centromere
.


Each chromatid consists of several
genes
.


Genes consists of a long
DNA

strand.


A string of
DNA

coiled around a few
histones

is called a
nucleosome
.


LOCATION OF DNA IN
A CELL

Locus
: Position of gene on chromosome

CHROMOSOME DUPLICATION
AND DISTRIBUTION

2. DNA STRUCTURE


DNA molecules are
polymers

called
polynucleotides.


Each polynucleotide is made of
monomers
called
nucleotides.


Each
nucleotide

consists of :


a
nitrogenous base
(Adenine,
Thymine, Cytosine or Guanine)


a
pentose sugar
(DNA =
Deoxyribose

sugar),



and a
phosphate group.


Nucleotide monomers are linked
together to build a
polynucleotide.


Adjacent nucleotides are joined by
covalent bonds
that form between the

OH group on the 3’ carbon
of one
nucleotide and the
phosphate on the 5’
carbon
on the next nucleotide.


These links create a
backbone of sugar
-
phosphate
units with
nitrogenous bases
as appendages.


The sequence of bases along a DNA
polymer is unique for each gene.


A polynucleotide and a single nucleotide

The different nitrogenous bases in DNA

Single ring structure

Double ring structure


A DNA molecule has
two
polynucleo
-
tides
spiralling around an imaginary axis,
forming a
double helix.


In the DNA double helix, the two
backbones run in opposite 5’ → 3’
directions from each other, an
arrangement referred to as
antiparallel.


One DNA molecule includes many genes.


The nitrogenous bases in DNA pair up
and form
hydrogen bonds:
adenine (A)
always with thymine (T),
and
guanine
(G) always with cytosine (C).

A DNA double helix structure

3.

DISCOVERY OF THE
DNA STRUCTURE


Early in the 20th century, the
identifi
-
cation

of the molecules of inheritance
loomed as a major challenge to biologists.

The Search for the Genetic
Material: Scientific Inquiry


T. H. Morgan’s
group showed that
genes

are located on
chromosomes
, the 2
components of chromosomes are
DNA &
protein

which became the candidates for
the genetic material.


Key factor in determining the genetic
material was
choosing appropriate
experimental organisms
-

bacteria & the
viruses

that infect them were chosen.


Discovery of the genetic role of DNA began
with research by
Frederick Griffith
in 1928.


Griffith worked with
2 strains
of a
bacterium
,
1 pathogenic (S cells)
&
1
harmless (R cells)


Heat
-
killed

pathogenic
strain were mixed
with
living cells
of
harmless

strain and the
result = some
living cells
became
pathogenic.


This phenomenon was called
transformation
, now defined as a change
in genotype & phenotype due to
assimilation of foreign DNA.


More evidence for DNA as the genetic
material came from studies of viruses that
infect bacteria.


Such viruses, called
bacteriophages

(or
phages
), are widely used in molecular
genetics research.

Bacterial cell

Phage head

Tail sheath

Tail

fiber

DNA


1952:
A. Hershey & M. Chase
experiments showing that
DNA

is
the
genetic material
of T2 phage.


To determine the source of genetic
material in the phage, they designed
an experiment showing that
only 1 /
2 components

of T2 (
DNA or
protein
) enters an E. coli cell during
infection


They concluded that the injected
DNA

of the phage provides the
genetic information.


After most biologists became
convinced that DNA was the genetic
material, the challenge was to
determine how its structure accounts
for its role…


M Wilkins & R Franklin
used
X
-
ray
crystallography

to study molecular
structure.


Franklin produced a picture of the DNA
molecule using this technique.


A picture of the DNA molecule using
crystallography by Franklin.



Franklin’s X
-
ray crystallographic
images of DNA enabled Watson to
deduce:


that
DNA was helical


the
width of the helix


the
spacing of the nitrogenous
bases



Width

suggested that the DNA
molecule was made up of
2 strands,
forming a double helix


Representations of DNA molecule



Watson and Crick: built models of a
double helix to conform to the X
-
rays &
chemistry of DNA.


Franklin concluded there were
2
antiparallel sugar
-
P backbones
, with
the
N bases paired
in the molecule’s
interior.


But:
How did bases pair
? A
-
A?/A
-
T?/
A
-
C?/A
-
G?......



But:
How did bases pair
?


They worked it out by using the
following image:

Purine + purine: too wide

Pyrimidine + pyrimidine:
too narrow

Purine + pyrimidine: width
consistent with X
-
ray data


W & C noted that the pairing of the
Nitrogen bases was specific, dictated
by the base structures.



Adenine (A)

paired only with
thymine
(T),
&
guanine (G)
paired only with
cytosine (C)



The W
-
C model explains
Chargaff’s
rules

which states that; in any
organism the amount of
A = T
, & the
amount of
G = C


4. THE ROLE OF DNA


DNA is vital for all living beings


even
plants.


It is important for:



inheritance
,


coding for proteins
and


the
genetic instruction guide
for
life and its processes.


DNA holds the instructions for an
organism's or each cell’s development
and reproduction and ultimately death.


DNA can replicate itself.

NON
-
CODING DNA


Multicellular eukaryotes have many
introns
(non
-
coding DNA)
within genes
and
noncoding DNA between
genes.


The bulk of most eukaryotic genomes
consists of noncoding DNA sequences,
often described in the past as “
junk DNA



Much evidence indicates that noncoding
DNA plays
important roles in the cell.


Sequencing of the human genome reveals
that
98.5% does not code for proteins,
rRNAs
, or
tRNAs
.





About 24% of the human genome
codes for
introns and gene
-
related
regulatory sequences.


Intergenic

DNA

is noncoding DNA
found
between genes:


Pseudogenes

are former genes that
have accumulated mutations and are
nonfunctional


Repetitive DNA
is present in
multiple copies in the genome



5. DNA REPLICATION


Replication begins at special sites
called
origins of replication
, where
the 2 DNA strands separate, opening
up a replication “bubble” (eukaryotic
chromosome may have many origins
of replication.


The enzyme
helicase

unwinds the
parental double helix.


Single stranded binding protein
stabilizes the unwound template
strands.


A
replication fork
forms.


The enzyme:
Topoisomerase

breaks,
swivels and re
-
joins the parental DNA
ahead of the replication for, to
prevent over winding.


The unwind complimentary strands
now act as individual
template

for 2
new strands.


RNA nucleotides
are added to each
DNA template
by the enzyme RNA
primase

to form
RNA primers
on both
templates


The enzyme
DNA polymerase III
add
free DNA nucleotides to the RNA
primer 3’ carbon.


The free nucleotides bond with H
-
bonds to their complimentary bases
on the DNA templates.


Along one template strand of DNA,
the DNA polymerase synthesizes a
leading strand
continuously, moving
toward the replication fork.



To elongate the other new strand,
called the
lagging strand
, DNA
polymerase must work in the
direction
away from the replication
fork.


The lagging strand is synthesized as a
series of segments called
Okazaki
fragments.


Each fragment has an
RNA primer

and
added
DNA strand
.


All the fragments are then joined with
the help of the enzyme
DNA ligase.


DNA polymerase I
then removes RNA
primers and replaces it with DNA
nucleotides.

6. PROOFREADING AND
REPAIRING DNA


DNA polymerases
proofread newly made
DNA, replacing any incorrect nucleotides


In mismatch repair
of DNA,
repair
enzymes

correct errors in base pairing.


DNA damaged by chemicals, radioactive
emissions, X
-
rays, UV light, & certain
molecules (in cigarette smoke for example)


In
nucleotide excision repair
, a
nuclease

cuts out & replaces damaged stretches of
DNA.

7.BIOTECHNOLOGY
AND GENOMICS

DNA CLONING

BIOTECH PRODUCTS


DNA CLONING


Cloning
is the reproduction of genetically
identical copies of DNA, cells or
organisms through some asexual means.


DNA cloning can be done to produce
many identical copies of the same gene


for the purpose of
gene cloning.


When cloned genes are used to modify a
human, the process is called
gene
therapy
.



Otherwise
, the organisms are called
transgenic organisms


these
organisms today are used to produce
products desired by humans
.


A. Recombinant
DNA (
rDNA
) and
B.
polymerase
chain reaction (PCR)

are
two procedures that scientists can use
to clone DNA


CLONING OF AN ORGANISM


A. CLONING A HUMAN GENE

HOW IS INSULIN MADE BY DNA CLONING?

(A. RECOMBINANT DNA)


A large quantity of insulin are being produced
by
recombinant DNA technology
.


This process is as follows:


1.

DNA that codes for the production of
insulin

is removed from the chromosome of a
human pancreatic cell.


2.

Restriction enzymes
cut the gene from the
chromosome (isolating the gene for insulin)

I

Insulin gene


cut
out with restriction
enzyme

Isolated insulin gene

3.
A
plasmid

(acting as a vector/carrier of
new gene) is removed from the bacterium
and cut open with a
restriction enzyme
to
form
sticky ends

Plasmid removed from
bacterium

Cut by restriction
enzyme

plasmid

Nucleus

BACTERIUM CELL

Sticky ends


4.

Ligase

(enzyme) is added to join the
insulin gene

to the
plasmid

of the
bacterium cell
-

forming
recombinant
DNA.






5.

The
recombinant DNA
can then be
reinserted into the bacterium, the
bacterium will then produce more
insulin
, therefore
cloning the gene.


Insulin gene
placed in
plasmid

by enzyme
Ligase

( attached
to
sticky ends
)





6. When the
bacterium reproduces
it makes
the
insulin inserted
into the plasmid.


7. The bacteria are kept in huge
tenks

with
optimum pH, temperature and nutrient
values, where they multiply rapidly, producing
enormous amounts of insulin, this is then
purified and sold.

Recombinant DNA placed into bacterium cell

B. POLYMERASE CHAIN REACTION


PCR


Used in
genetic profiling
.


To solve crimes


criminals usually leave DNA
evidence at the scene of the crime in the form
of saliva, blood, skin, semen and hair. These all
contain DNA. If only a little bit of DNA is found
or the DNA is old, we can make
copies

of the
available DNA by means of
PCR.


From the DNA produced through
PCR
,
DNA
fingerprint
can be generated.

PCR

method


1. Sample containing DNA is
heated

in a
test tube to
separate DNA
into single
strands.


2.
Free nucleotides
are added to the test
tube with
DNA polymerase
(enzyme), to
allow DNA replication.


3. DNA is
cooled
to allow free nucleotides
to
form

a
complementary strand
along side
each single strand.


4. In this way the
DNA is doubled
giving
sufficient amount of DNA to work with.


DNA SCREENING AND FINGERPRINT
TECHNIQUE


1. Sample of
DNA
is cut into
fragments

by means of
restriction enzymes.


2. Negative charged electrode at one end of a
rectangular flat piece of gel and a positive
electrode is placed at the other end.


3. The
DNA is placed at the negative
end of the gel
and starts to move to the positive end.
Smaller
fragments move faster
than the larger ones.
Separation occurs on the basis of size. This process
is called
gel electrophoresis
.



4. DNA is then pressed flat against the gel and
transferred to
filter paper
.


5.
Radioactive probes
bind to special DNA
fragments.


6.
X
-
rays

are taken of the filter paper. The DNA
probes show up as dark bands on the film. The
pattern of these bands is the
DNA fingerprint
.

DNA fingerprinting


GEL ELECTROPHORESIS


DNA FINGERPRINT


BIOTECHNOLOGY PRODUCTS


Today transgenic bacteria, plants and animals
are called genetically modified organisms
(
GMO’s
).


The products that
GMO’s

produce are called
biotechnology products.


GENETICALLY MODIFIED BACTERIA


Recombinant DNA
is used to make transgenic
bacteria.


They are used to make
insulin, clotting factor
VIII, human growth hormone
and
hepatitis B
vaccine.


Transgenic bacteria is used to protect the
roots of plants from insect attack, by
producing insect toxins.

GENETICALLY MODIFIED PLANTS


Example =
pomato


Genetically modified to produce potato's
below the ground and tomato's above the
ground.


Foreign genes
transferred to cotton, corn, and
potato strains have made these plants
resistant to pests
because their cells now
produce an insect toxin.


Read p. 253 for more examples

GENETICALLY MODIFIED ANIMALS