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• Genetic Variation = refers collectively to the
diversity of genomes that give rise to different
individuals
– includes small differences between genomes of
members of the same species and greater differences
between the genomes of different species
– the ultimate source is changes in DNA (mutations) that
alter its nucleotide sequences
• some small due to rare “mistakes” in DNA replication and
repair
• some large, due to different processes including DNA
recombination, the activities of viruses, and mobile genetic
elements.
– Also sexual reproduction results in re-assortment of the
gene pool into new combinations in different
individuals
Division of bacterial cells
takes about 20-25 minutes.
With no mistakes, each
parent gives rise to
identical daughters
Can be grown in liquid
medium with nutrients and
oxygen at around 37˚C
Grown on solid surface of agar, colonies develop from
individual bacteria if the original suspension is dilute, and
spread over the surface of the agar.
Individual colonies
can be selected for
study.
Mutations in bacteria can be selected by a change in the environment. Grown
in the presence of rifampicin, mutants with an RNA polymerase that is not
disabled by this antibiotic survive and take over the culture. In any large
population of bacteria there will be many different “mutants” (genetic variants).
These can be selected for during a change in the environment (natural
selection).
Bacterial genes can be
acquired from other
bacteria by several
different routes.
Direct cell to cell
transfer during
bacterial mating or
conjugation is shown
here.
Quesstions 2,3,5,6,
8,9, 11,13,14,17
An example of DNA
exchange:
E. Coli strain with
mutation making it
unable to make
methionine grown
with E. Coli strain
with mutation making
it unable to make
leucine results in the
appearance of a new
strain able to make
both. How?
DNA recombination
Bacterial mating occurs between a bacterium with an F
plasmid and one without one. A cytoplasmic bridge is
formed, DNA is duplicated and transferred.
1.
2.
Plasmids are small, circular, double-stranded DNA separate from the bacterial
chromosome which can replicate itself. F plasmids (fertility) contain the genes needed
for conjugation.
Usually only a small number
of genes on the plasmid are
transferred.
Rolling circle
replication
Transfer of bacterial
chromosomal genes by an F
plasmid after integration into
the bacterial chromosome.
Widespread use of antibiotics
leads to many clinical
isolates that are resistant to
the antibiotic typically used –
as in Neisseria gonorrhoeae
to penicillin. These contain
plasmids that encode proteins
that inactivate the antibiotics
Bacteria can take up the DNA from their surroundings - Bacterial
Transformation.
Uptake of naked DNA is active and receptormediated and has become an important tool in
the laboratory for research and biotechnology.
Once inside of a bacteria,
naked DNA will only survive
and be passed on to progeny if
it is incorporated into the
bacterial chromosome.
Homologous Recombination
occurs in all organisms and
takes place between two DNA
molecules with similar
sequences.
DNA aligns so that
base pairs of
homologous
sequences match
complementarily
Site of exchange
can occur
anywhere in this
region
Cleavage of the
DNA molecules
and rejoin is
precise, no
nucleotides are
lost or gained
Cross-strand
exchange or
Holliday
junction
Homologous recombination can lead either to an exchange
of DNA between two molecules or to the combination of
two circular DNA molecules into one.
F plasmid contains a short homologous stretch of DNA
Cells utilize specialized proteins to facilitate homologous
recombination; these proteins nick DNA, catalyze strand
exchange, and cleave Holliday structures.
Genes can be transferred
between bacteria by bacterial
viruses - bacteriophages.
This is a simple virus - simpler
than any real virus.
Consists of a small doublestranded DNA moleculs
must enter the cell, replicate its
DNA, and transcribe some,
especially the protein coat.
Viral DNA and protein coat
spontaneously assemble.
Bacteriophage
lambda
Latent state
Prophage
lies dorman
Damage to
bacteria,
ultraviolet
light
Encodes 50 - 60 proteins
Lytic infection
Integrates by Site-specific recombination
Virus encodes a specialized enzyme - lambda
integrase - which recognizes a specific sequence
of bacterial DNA and a specific sequence of viral
DNA. It binds these and then brings them
together, breaks both, and then rejoins them
Exits by doing the same thing in reverse.
Excises itself inaccurately on occasion
The modified lambda infects a new host. Two
ways the bacterial DNA is transferred to another
bacteria.
1. Integration
2. Homologous recombination (transduction)
Homologous recombination can lead either to an exchange
of DNA between two molecules or to the combination of
two circular DNA molecules into one.
Transposable Elements (transposons) create more genetic diversity.
Range from several hundred to tens of thousands of base pairs. Typical
lab E. coli contains 10-20 different transposons, with many having
multiple copies. Transposons move within a DNA molecule by using a
special recombination enzymes - transposases - encoded by the
transposon. Sequences in red are recognized only by the particular
transposase encoded. Transposons can
“pick up” host DNA by
homologous recombination between two
identical transposons.This probably
led to the antibiotic resistant
plasmids and transposons.
Transposons provide a number of advantages: 1. Some contain transcriptional
promoters, land near a bacterial gene, and cause it to be expressed at a different level or
under different controls. 2. Some are present in multiple copies, allowing
rearrangement by homologous recombination between transposons. 3. Some contain
drug resistant genes which then can jump into plasmids and be transmitted to other
bacteria
Donor DNA and targe
can be on the same
molecule or on different
molecules (bacterial
genome + plasmid)
Generally a transposon
can use only one of
these methods.
• DNA replication is remarkably accurate.
• However, genomes are in a state of slow
but constant change
– mutations accumulate over time
– genes are transferred from one bacterium to
another
– viral genomes move in and out
– transposons change position
• Most alterations in the genome are
harmful, but, when the environment
changes (antibiotics are present) genetic
variation is crucial for survival.
• Sources of Genetic change in Eucaryotic
Genomes
• Bacterial genomes are streamlined, genes are closely
packed with relatively little spacer DNA and few introns
• Mammalian genome contains enormous amounts of nongene DNA including introns, spacer DNA between genes
and various types of repetitive DNA sequences with
similarities to transposons. Also a large amount of gene
duplication, leading to the large families of highly related
genes and different forms of each gene - alleles.
– this DNA has far-reaching effects
– alleles increase the “gene pool” - the entire collection of
alleles of every gene present in a species
Random DNA duplications
create families of related
genes.
Ex. Different forms of actin expressed in
different types of muscle cells and in other
cell types.
There are 5 beta-globin genes, each
produced at a different time during
embryonic, fetal, and adult development.
Each with a different oxygen-binding and
-releasing characteristic appropriate for
the time it is expressed. And each under
independent regulation.
Comparing the sequences of genes like the
beta-globin gene leads to models like this
which may reflect the evolution of the
gene through thousands of years.
Gene duplication by unequal crossing-over.
Genes encoding new proteins can be created by recombination of exons,
without damaging the exon, due to the
presence of introns.
New mRNA can be spliced correctly due to the
presence of the proper intron sequences.
• The presence of introns greatly increases
– the probability that DNA duplications
will give rise to functional genes
– and the probability that a chance
recombination event can generate a
functional hybrid gene by joining two
initially separate exons coding for
different protein domains – exon
shuffling.
Some Results of exon shuffling through time.
A large part of the DNA of multi-cellular eucaryotes
consists of repeated, non-coding sequences
• About 70% in humans is “unique” DNA coding for
proteins or RNA and including intron DNA
• Remaining 30% includes two types of repeated, noncoding sequences
– Satellite DNA = highly repeated short sequences that form serial
arrays (about 1/3), which can be clustered at the centromeres and at
the ends of chromosomes.
• Function is unknown and it varies greatly between individuals
of the same species
– Repetitive sequence DNA composed of complex repeated
sequences interspersed throughout the genome derived from a few
types of transposable DNA sequences like those in bacteria
• DNA in humans and primates is unusual in that it contains a
remarkable number of two of these transpoable DNA sequences.
About 10% of the human genome consists of two families of
transposable sequences. Some move using the cut-and-past mechanism
in Figure 18A. Others move via an RNA intermediate =
retrotransponons – perhaps unique to eucaryotes.
L1 transposable element, or LINE-1 = 4% of the
total mass of the human DNA
The reverse transcriptase is encoded in L1.
Alu sequence is shorter and is about 5%. It does
not code for a reverse transcriptase – requires one
encoded elsewhere. Many are non-movable
Evolution of genomes has been accelerated by transposable elements.
They are a significant source of mutation
Insertion into coding sequences of a gene or into its regulatory region is relatively frequent cause
of spontaneous mutations in some organisms.
Ex. Some mutations in Factor VIII (hemophilia) result from insertion of transposable elements
into the gene.
They provide opportunities for genome rearrangements by serving as target of homologous
recombination.
Exon shuffling caused by a transposon. Transposase uses the beginning
of the 1st element and the end of the 2nd element to replicate and move
the segment, including exon 2A into a different gene.
DNA rearrangement caused by a transposable element can
produce a dramatic change in the organism.
normal
Legs where antennae should be
Lytic effect of virus
Genomes of viruses can vary greatly.
The amount of DNA or RNA that can be packaged is limited.
Viral genomes encode few proteins and rely on host enzymes
to replicate and translate their genes, including viral coat
proteins and proteins that attract host enzymes to replicate
their genome.
Viruses are parasites that can reproduce
themselves only inside a living cell.
This is the life cycle of a singlestranded RNA virus. RNA viruses must
encode an RNA replicase, to replicate
its genome. Negative stranded RNA
viruses must carry an RNA replicase in
their protein coat.
Viruses are parasites that can
reproduce themselves only
inside a living cell. The
smallest viruses contain 3
genes. protein coat is made
up primarily of one
polypeptide. More complex
viruses have genomes up of
up to several hundred genes
and are surrounded by an
elaborate shell composed of
many different proteins.
HIV is a retrovirus
Host
V
Latent = problem for
treatment
V Integrase
Since reverse transcriptase is not used by host cells it is a prime target of
drug development
Retroviruses may
have derived from
retrotransponsons that
long ago acquired
additional genes
encoding coat proteins
etc.
Viruses that integrate into host cell DNA are, like
transposable elements, potential agents of genetic
change.
About 10% of the human genome consists of two families of
transposable sequences. Some move using the cut-and-past mechanism
in Figure 18A. Others move via an RNA intermediate =
retrotransponons – perhaps unique to eucaryotes.
Retroviruses can pick up host genes. This can lead to cancer.
The virus is then known as a tumor virus.
Ex. Rous sarcoma virus picked up a gene from chickens that
encodes a protein kinase important in control of cell division.
This gene was altered in the virus to become hyperactive.
Expression of the viral src gene (an oncogene) leads to the loss
of control of cell division - cancer.
Egg with sperm
bound
Sexual
reproduction is
costly in terms of
resources spent
but is
advantageous in
terms of genetic
variation created
and changing
environments.
Genetic
variation
Genetic
variation
chiasmata
Reassortment =
genetic variation
23 possibilities
2n possibilities
Ura + bacteria can grow on medium that lacks the base uracil whereas mutant Ura – bacteria
cannot. Ura – bacteria can, however, grow on medium that contains urabegone, a drug that kills
Ura + cells. You inoculate a Ura + bacterium into media containing uracil and allow it to divide
until there are 10 9 cells, which you then dilute and spread onto plates containing urabegone
and uracil. You get 50 colonies in all. Which of the following statements are likely to be true?
A. All of the cells in a given urabegone-resistant colony have the same mutation
in a gene required for growth in the absence of uracil.
B. The cells in all 50 of the urabegone-resistant colonies all have the same mutation
in a gene required for growth in the absence of uracil.
C. All of the Ura + cells that did not grow on the urabegone plates were genetically
identical.
D. If you inoculate a Ura + bacterium and a Ura – bacterium together into media
containing uracil and grow them for a day, the Ura – bacteria will compose only
a small fraction of the population.
E. If you inoculate a Ura + bacterium and a Ura – bacterium together into media
containing uracil and grow them for a day, the Ura – bacteria will significantly
outnumber the Ura + population.
The Ura – bacteria described in Question 9–3 differ from the Ura + bacteria in that:
A. the Ura + bacteria do not use uracil in their cells.
B. the Ura – bacteria lack an enzyme required to synthesize uracil.
C. the Ura – bacteria lack an enzyme required to synthesize nucleotides from
bases.
D. the Ura – bacteria lack a transport protein required to take up uracil from the
medium.
E. the Ura – bacteria lack an enzyme that removes uracil nucleotides from DNA.
What are you likely to find if you inoculate a bacterium from a Ura – colony described in
Question 9–3 into medium containing uracil and then plate out 10 12 of the resulting cells onto
media lacking uracil? Explain your answer.