Download Gene Mutations

Cytology of Genetics
 Organisms with circular DNA
 Organisms with linear DNA
Organisms with circular DNA Chpt. 8
 virus/phage
 bacteria
Advantages of circular DNA
 no problems with replication of the ends of
the DNA
 protection from exonucleases
1
Two ways to replicate circular DNA
 bi-directional - produces two complete
circular pieces of DNA, good when it is
desired to have a limited number of copies
of the DNA at one time. Example bacterial replication
 rolling circle - produces single or double
stranded linear DNA, good when linear
pieces of DNA are required or when a high
number of copies of DNA are needed.
Examples - viral replication and bacterial
conjugation.
2
Virus/Phage Replication Chpt. 8 pgs 215-230
Virus - an ultramicroscopic, obligate,
intracellular parasite incapable of
autonomous replication.
Virus can be separated by the way they store
their genetic information.
 Double-stranded DNA virus
 Single-stranded DNA virus
 Single stranded RNA virus
3
Double-stranded DNA virus
1. DNA is stored in the virus particle in linear
form.
2. After injection it forms circular DNA aided
by ‘sticky ends’ at the ends of the linear
DNA. ‘Sticky ends’ refer to short single
stranded sections at the ends of the DNA
that are complementary.
3. The virus genes are then transcribed.
4. The virus genome is then replicated,
probably by bi-directional replication first
then by rolling circle when linear DNA is
needed for packaging in the newly formed
virus particles.
4
Single-stranded DNA virus
1. Single strand of linear DNA is injected into
the cell by the virus particle. This strand is
the ‘sense’ strand for transcription and is
called the ‘+’ strand.
2. The initial strand is replicated to produce a
‘-‘ strand resulting in a doubled -stranded
DNA molecule that can then form circular
DNA.
3. Rolling circle replication then occurs using
the ‘-‘ strand as the template to produce
multiple copies of linear ‘+’ strands. These
‘+’ strands are then packaged into the viral
particles.
5
Single-stranded RNA virus replication
How do RNA viruses replicate? Since the
cells the virus invades do not have the
enzymes for RNA replication, the virus must
carry the genetic information for the enzymes
necessary for their replication.
 RNA  RNA
 RNA  DNA  RNA
RNA  RNA virus replication
This type of virus carries an enzyme called
RNA replicase that will replicated RNA.
Because it is single-stranded, replication of
RNA RNA virus is similar to that of singlestranded DNA virus without the circular
DNA step.
6
1. Linear RNA (can be in multiple pieces) is
injected into a host cell. Since it is ready to
be translated it is called the ‘+’ strand.
2. The RNA is translated to produce
replicase or in the case of eukaryotes the
virus carries in a replicase enzyme with the
RNA. This produces a ‘-‘ strand.
In eukaryotes replicase is carried in the
virus particle, why would this be
important in eukaryotes?
3. The replicase then uses the ‘-‘ stand to
produce copies of the ‘+’ strand.
4. The ‘+’ RNA strands are then packaged
into the viral particles.
- Why can there be so much variation in
RNA viruses (or why so many strains of
the flu virus?
7
Why do RNA viruses vary so much in their
proteins (or why do you need a flu shot every
year?)
RNA  DNA  RNA virus replication
This virus is sometimes called a retrovirus. It
carries the enzyme reverse transcriptase that
allows it to synthesize a strand of DNA using
RNA as the template. This type of virus may
also incorporate into the host’s DNA once it is
in DNA form.
So the replication goes against the central
dogma of genetics.
DNA

RNA

RNA
DNA


protein
RNA

protein
8
In DNA form can incorporate into the host
DNA.
In RNA form can be packaged into new virus
particles
1. The linear RNA enters the host cell and is
translated to produce reverse transcriptase.
The reverse transcriptase then uses the
mRNA as a template to produce singlestranded DNA.
2. The host cell’s DNA polymerase then
replicates a complementary strand of DNA
producing double-stranded DNA.
3. The viral DNA can now integrate into the
host’s DNA.
4. The viral DNA is now replicated with the
host’s DNA and all subsequently produced
cells carry the virus. If the virus infects a
9
germ cell it can be passed on to the next
generation through the gametes.
5. The viral DNA can be transcribed while it
is in the host DNA to produce new viral
particles.
Examples of retroviruses
HIV, feline leukemia virus, sarcoma virus
How would you stop a retrovirus?
How could you cure an individual infected
with a retrovirus?
10
Recombination in virus/phage
 requires simultaneous co-infection
 replication of the DNA or RNA
 copy-choice error in replication
example: replicase moving from one RNA
strand to another RNA strand during
replication.
 complementary pairing and recombination
between the two DNA molecules resulting
in an exchange of DNA sequences. Viral
particles carrying the DNA hybrids are
called recombinants.
11
Bacteria/Prokaryotic Replication Chpt 8
Prokaryotes - all microorganisms that lack a
membrane bound nucleus containing
chromosomes.
Bacteria - have genetic information in two
forms:
1. Bacterial chromosome where the
majority of the bacterial genes are
stored. The DNA is circular and
approximately 107 nucleotide bases.
There is usually one copy of the
bacterial chromosome per cell.
12
2. plasmids are small circular pieces of
self-replicating DNA that may contain
genes for one to several enzymes.
These genes may supply a necessary
trait such as antibiotic resistance to the
bacteria.
There may be several different
plasmids in a cell and they may differ
in number in a cell.
Plasmids may or may not be
transferred during cell division.
13
Bacterial replication:
1. Replication of the bacterial DNA (including
plasmids) by bi-directional replication.
2. Division of the cells by binary fission.
3. Genetic information should be identical
between the daughter cells unless there are
genes on a plasmid.
4. Only way to make a genetic change is by
recombination between two unrelated
bacteria.
14
Bacterial Recombination Chapter 8
Bacterial recombination is the exchange of
genetic material (DNA) between cells. It can
occur three ways:
1. transformation - free-floating DNA
2. conjugation - direct contact between cells
3. transduction - uses a vector
Transformation - Genetic modification of
bacteria by incorporation of free-floating
foreign DNA. (Griffith/ Avery, MacLeod and
McCarty experiments)
15
Conjugation - ‘Bacterial sex’ - The exchange
of genetic information by the union of two
bacterial cells resulting in the transfer of
DNA from a donor cell (+) to a recipient cell
(-).
 Transfer of the genetic information is
controlled by and due to the presence of a F
(fertility) factor plasmid in the donor cell.
 F factor: circular double stranded DNA
plasmid with 104 nucleotide base pairs.
 Function of the F factor is to allow a
physical connection to be made between
donor (+) cells and recipient (-) cells.
16
 The F factor can exist in three forms in the
cell
 independent plasmid (cell type F+)
 incorporated in the bacterial
chromosome (cell type Hfr)
 independent plasmid but carrying part of
the bacterial chromosome (cell type F’)
Only the last two forms of the F factor
plasmid can cause genetic recombination with
bacterial chromosome genes.
17
When the F factor plasmid is incorporated
into the bacterial chromosome it is possible to
have a high frequency of recombination
(Hfr).
The reason for this is that when conjugation
occurs the plasmid pushes the bacterial
chromosome through first, transferring a
copy of the donor bacteria genes to the
recipient cell.
Hfr strains of bacteria that have the F factor
plasmid inserted can be used to map the gene
order of the bacterial chromosome. Which
genes are transferred first is dependent on the
site of integration of the F factor plasmid and
its orientation in the bacterial chromosome.
18
To study gene order the donor and recipient
bacteria must differ genetically, i.e. have
differential expression for antibiotic
resistance, amino acid synthesis etc.
For mapping allow conjugation to occur
between the two bacteria, disrupt
conjugation at various times, then test to
determine which genes have been transferred.
Genes with the highest level of recombination
are closest to the insertion site of the F factor.
Gene order differs among Hfr strains of
bacteria because of the variation in insertion
sites and orientation of the F factor in the
bacterial chromosome.
19
Mapping the bacterial chromosome using Hfr
conjugation.
 Use Hfr strains with different insertion sites
and orientations to map the entire bacterial
chromosome.
 Donor and recipient cells must differ for a
set of ‘marker’ genes.
 Marker genes allow the researcher to
observe when recombination has occurred.
Example:
Hfr strain
A37
A40
B39
B50
C21
earliest gene (1st out)-----------last gene out
arg
trp
gal
lac
pro
cys
thr
pro
lac
gal
pro
lac
gal
trp
arg
thr
cys
mal
arg
trp
gal
trp
arg
mal
cys
20
When the F factor disassociates from the
bacterial chromosome incorrectly in that it takes
part of the bacterial chromosome DNA with it,
you have a special form of conjugation called
‘sexduction’ (F prime or F’ ).
With this situation, whenever conjugation
occurs the bacterial chromosome genes on the
F factor plasmid get transferred every
conjugation event resulting in an apparent
high rate of recombination for one or a few
genes.
21
Transduction - the transfer of bacterial
genetic material from one bacterium to
another using a virus/phage as a vector.
There are two forms of transduction:
1. specific or specialized
2. general
Specific or specialized transduction occurs
when only specific genes are transferred by
the phage.
It occurs with temperate phage that can exist
as a self-replicating unit or integrated into the
host DNA. When the virus integrates into the
host DNA it inserts into only one site.
22
When it disassociates incorrectly it can take
the bacterial gene on either side of the
integration site by accident.
When one of these ‘defective’ phage infects a
cell, it transfers the bacterial gene into the
new host.
Only the genes adjacent to the insertion site
can be transferred, hence the name specific or
specialized transduction.
23
General transduction occurs when a piece of
the bacterial chromosome is accidentally
packaged into the phage particle instead of
the phage DNA
When this phage infects a cell the bacterial
DNA is injected into the cell and may
recombine with the host DNA.
24
Summary of bacterial recombination
1. transformation - where foreign DNA is
picked up from the environment by the cell.
2. conjugation - where physical contact
between two cells is required for the
transfer of DNA.
3. transduction - where a vector is required to
transfer the DNA between cells.
25