Download Effects of mutations

Microbial Genetics
Unlocking the Secrets of Heredity
Chapter 8
Chromosomes
•Chromosome: discrete cellular structure composed of a neatly
packaged DNA molecule
•Eukaryotic chromosomes
- DNA wound around histones
- located in the nucleus
- diploid (in pairs) or haploid (single)
- linear appearance
•Prokaryotic chromosomes
- DNA condensed into a packet by means of histone-like
proteins
- single, circular chromosome
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A sampling of genes related to
obesity in the human genome
• http://www.obesity.chair.ulaval.ca/genemap.ht
ml
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• A closer look at
chromosome 1
• Genes are
SPECIFIC &
DISCRETE
segments of
DNA
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• A map of E. coli’s
~5000 genes
• Notice it is single &
circular
• Does E. coli have 1 or
2 alleles of each
gene? How do you
know?
• Humans were first
thought to function
with 100,000 genes
and now the number
has dropped to
~35,000 genes
although this is still a
hot topic in research
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DNA is lengthy and occupies a small part of the cell by coiling up
into a smaller package.
Fig. 9.3 An Escherichia coli cell disrupted to release its
DNA molecule.
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Genome
• Genome: sum total of genetic material of an organism
- most of the genome exists in the form of
chromosomes
- some appears as plasmids or in certain organelles of
eukaryotes
- genome of cells composed entirely of DNA
- genome of viruses can contain either DNA or RNA
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Gene
•Gene
-
a certain segment of DNA that contains the necessary
code to make a protein or RNA molecule
•Three categories of genes
- structural genes: code for proteins
-
genes that code for RNA machinery used in protein
production
-
regulatory genes: control gene expression
Genetic Terms
• Genotype
• an organism’s genetic makeup; its entire
complement of DNA
• Phenotype
• is the expression of the genes: the proteins of the
cell and the properties they confer on the
organism.
• Size, shape, color, environment
• Which one is easier to see?
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The DNA Code
Hydrogen bond
H
H
N
N
• Nucleotide: basic unit of DNA
structure
• phosphate
• deoxyribose sugar
• nitrogenous base
N
N–H
G
C
H
N
N
O
N–H
Sugar
3′ OH
H
P
D
5′
4′
D
1′
5′
P
2′
P
D
3′
C
G
O
P
D
O
D
P
P
A
D
O
P
D
O
T
P
O
C
G
O
D
O
P
• Nucleotides covalently bond to each
other in a sugar-phosphate linkage
P
C
G
D
D
P
P
T
D
A
D
O
P
• Pairing of bases dictated by the
formation of hydrogen bonds
between bases
H
H–N
O
O
D
P
O
C
G
O
D
O
P
P
P
T
A
D
D
P
5′
D
D
3′
5′
H
OH
H
N–H
N
N
N
O
H– N T
(a)
H
N
N
Sugar
CH3
H
O
Nature of the double helix
- antiparallel arrangement: one side of the
helix runs in the opposite direction of the
other
- the order of the bond between carbon on
deoxyribose and the phosphate is used to
keep track of the direction of the two sides
- one side runs from 5’ to 3,’ and the other
side runs 3’ to 5’
- this is a significant factor in DNA synthesis
and protein production
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DNA Replication
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DNA Replication
• What you need to replicate DNA:
1. Original DNA template (parental chromosome)
2. Nucleotides (Guanine, Cytosine, Adenine,
Thymine)
a pool of nucleotides will be free floating in the
cytoplasm waiting to be used
3. Enzymes
I.e., DNA polymerase (hooks together nucleotides),
ligase (ligates)
4. Energy (ATP)
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Replication
• Don’t worry about knowing the leading
strand, lagging strand and 5’ and 3’
terminology for the exam. These are more
in depth than we will go.
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DNA Replication in Prokaryotes
• Certain enzymes
unwind the DNA.
• Then, DNA
polymerase can
read the parent
strand and attach a
complementary
nucleotide to the
new strand of DNA.
• The nucleotides are
free in the
cytoplasm.
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DNA replication is semi-conservative since each new
chromosome will have one “old” and one “new” strand
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Transcription
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Transcription (RNA Synthesis)
• What you need to synthesize RNA:
1. Original DNA template (parental chromosome) with a
promoter site (DNA sequence indicating start site) and a
terminator site
2. Nucleotides(G, C, A, U)
Ribose (sugar) + phosphate + N base
Uracil is substituted for thymine
3. Enzymes
I.e., RNA polymerase (hooks together nucleotides)
4. Energy (ATP)
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Transcription
• RNA polymerase: large, complex enzyme
that directs the conversion of DNA into RNA
• Template strand: only one strand of DNA
that contains meaningful instructions for
synthesis of a functioning polypeptide
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Transcription
4 types of RNA can be transcribed:
1. Messenger ribose nucleic acid (mRNA)
mRNA (RNA molecule that serves as a message of the
protein to be produced)
2. Transfer ribose nucleic acid (tRNA)
tRNA (64 different tRNA molecules participate in
translation)
3. Ribosomal ribose nucleic acid (rRNA)
rRNA (forms the ribosome)
4. Regulatory RNA
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The RNAs
•RNA is similar to DNA in terms of its general properties, but its
structure is different in several ways
- single-stranded molecule that exists in helical
form; can assume secondary and tertiary levels
of
complexity, leading to specialized forms of RNA (tRNA
and rRNA)
-
contains uracil (U) instead of thymine; does not
change the DNA code because uracil still follows the
pairing rules
-
contains ribose rather than deoxyribose
The RNAs
•
•
•
•
mRNA- message from DNA, single stranded
rRNA- part of ribosome
tRNA- transfers amino acids to ribosome
Regulatory RNAs:
- micro RNAs, anti-sense RNAs, riboswitches,
small interfering RNAs
• Primer RNAs: operative in both prokaryotic and
eukaryotic cells
• Ribozymes: remove unneeded sequences from
other RNAs
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Transcription: Initiation
• RNA polymerase recognizes promoter region
• RNA polymerase begins its transcription at a special
codon called the initiation codon
• As the DNA helix unwinds it moves down the DNA
synthesizing RNA molecule
Transcription: Elongation
Direction of
transcription
Early mRNA
transcript
Nucleotide
pool
• During elongation the mRNA is built, which proceeds
in the 5’ to 3’
direction (with regard to the growing RNA molecule)
• the mRNA is assembled by the adding nucleotides
that are complementary to the DNA template.
• As elongation continues, the part of DNA already
transcribed is rewound into its original helical form.
Transcription: Termination
Elongation
Late mRNA
transcript
At termination the polymerases recognize
another code that signals the separation
and release of the mRNA strand,
or transcript.
Translation
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Translation
• Decoding the “language” of nucleotides
and converting/translating that
information into the “language” of
proteins.
• The nucleic acid “language” is in the
form of codons, groups of three mRNA
nucleotides.
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Where does translation occur?
• Translation occurs at the
RIBOSOME!
• The green mRNA strand is
“threaded” through the
ribosome.
• The ribosome “reads” the
mRNA strand codons with
the help of the genetic
code and tRNA (next
slides)
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tRNA
• Decoder molecule which
serves as a link to translate
the RNA language into
protein language
– One site of the tRNA has an
anticodon which
complements the codon of
mRNA
– The other site of the tRNA
has an amino acid
attachment site
corresponding to a specific
amino acid as noted in the
genetic code
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Codons
• Triplet code that specifies a given amino
acid
• Multiple codes for one amino acid
– Degenerate(repetitive) code is good to allow for
a certain amount of mutation to occur without
having an effect on the amino acid sequence
– 1 Start codon
– 3 Stop codons
– 64 total possible codons
– 20 amino acids
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Translation and the “Genetic Code”
• We use the “genetic code”
(at right) to translate mRNA
nucleotide sequence
(codons) into amino acid
sequence which make up
proteins.
•
The “genetic code” is
degenerate which allows
for a certain amount of
mutation. I.e. UUU and
UUC both code for Phe
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Translation and the “Genetic Code”
• There is one start codon,
AUG, that codes for the
amino acid methionine.
•
There are 3 stop codons,
UAA, UAG and UGA that
signal the ribosome to stop
translation and let go of the
polypeptide chain (protein).
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Translation
• Ribosomes
bind mRNA
near the start
codon (ex.
AUG)
• tRNA anticodon
with attached
amino acid
binds to the
start codon
Translation
• Ribosomes move to the
next codon, allowing a
new tRNA to bind and
add another amino acid
Translation
• Series of amino acids
form peptide bonds
Translation
• Stop codon terminates
translation
Polyribosomal Complex
- a single mRNA is long enough
to be fed through more than one
ribosome
- permits the synthesis of
hundreds of protein molecules
from the same mRNA transcript
- occurs only in prokaryotes,
where there transcription and
translation both occur in the
cytoplasm
- Would you see this in
Eukaryotes?
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Introns and Exons
Eukaryotic mRNAs
code for just one
protein, unlike
bacterial mRNAs,
which often contain
information from
several genes in
series
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Transcription and translation
in eucaryotes
• Similar to procaryotes except
– AUG encodes for a different form of methionine
– Transcription and translation are not simultaneous
(since eucaryotes have a nucleus----transcription
occurs in the nucleus, translation occurs ?)
– Eucaryotes must splice out introns to achieve a
mature mRNA strand ready to go to the ribosome.
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How are genes regulated?
• Cells regulate genes in 3 major ways:
1. Feedback inhibition
– The end-product inhibits the pathway (similar to a
thermostat….when it reaches the desired temperature it turns
off)
2. Enzyme induction
– If a substrate is present, the enzyme for the substrate is
induced.
3. Enzyme repression
a. If a nutrient is present, the enzyme to make it is repressed.
b. If a nutrient is absent, the enzyme to make it is turned on.
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Operons
-
only found in bacteria
coordinated set of genes
all regulated as a single unit
either inducible or repressible
lac Operon
lac Operon
Phase Variation
• Bacteria turn on or off a complement of
genes that leads to obvious phenotypic
changes
• Phenotype is heritable!
• Most often traits affecting the bacterial cell
surface
• Examples:
- Neisseria gonorrhoeae: production of attachment
fimbriae
- Streptococcus pneumoniae: production of a
capsule
What if a gene changes?
• Mutation=a change in the sequence of DNA
• Effects of mutations
• none-->no change in a.a. sequence or….
• Good-->new aa. Seq-->antibiotic resistance
– Increases variability in the gene pool
• Bad-->new aa. Seq-->mutate active site of enzyme
• For humans, cancer is the product of a combo of bad
mutations.
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Types of Mutations
• Point Mutation
• put the cat out--->puc the cat out
• put the cat out--->put
• Frameshift (reading frame of mRNA shifts)
•
•
•
•
put the cat out--->put hec ato ut
Deletion
Addition
Duplication
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The Effects of Base Substitution
(a point mutation)
• When a base is substituted in DNA
the mutation may have 2 effects:
– Changes the amino acid
– Does not change the amino acid
– Why doesn’t a mutation always change
the amino acid sequence? Because the
genetic code is degenerate and has
amino acids that may be coded for by
different codons. (I.e., AAA and AAG
both code for phenylalanine)
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The Effects of Frameshift Mutations
• The addition,
deletion or insertion
of one or more
nucleotides
drastically changes
the amino acid
sequence.
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Mutation Rates
• Normal Mutation Rate=1/1 million per gene
– Mutations are constantly occurring since our
enzymes are not 100% perfect …These are
called spontaneous mutations and increase in
occurrence as we age….when do we get
cancer?
• Mutagen=certain chemicals or radiation that
bring about mutations.
• Mutagen Mutation Rate= 1/1000-1/100,000
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per gene (10-1000X the normal rate)
Repair of mutations involves enzymes recognizing, removing, and
replacing the bases.
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Mutagen Examples
• 5-Bromouracil and acridine
are 2 mutagen examples
that can “insert”
themselves in DNA and
cause errors in DNA
replication, transcription
and translation.
• Notice how similar in
structure mutagens can be.
There is just one change to
thymine that can have dire
consequences
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Thymine Dimers Caused by
Radiation
• Radiation, such as X-rays
and UV rays, can cause
dimers to form in DNA.
• Thymine dimers can
interfere with DNA
replication, transcription
and translation.
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What is the connection to
cancer?
• Cancer is a genetic disease. It is the
consequence of a change in DNA
sequence.
• Carcinogen=substance that causes
cancer
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What is the connection to
cancer?
• Are mutagens also carcinogens?
• The Ames Test uses bacteria to identify
possible carcinogens by looking for
mutations to occur. Once a mutagen is
identified, it is tested in animals to test if
it is a carcinogen.
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The Ames test is used to screen environmental and dietary
chemicals for mutagenicity and carcinogenicity without using
animal studies.
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Is there another way for the
genetic makeup to change?
• Yes-->Genetic Recombination
• Effects of genetic recombination
– increase diversity in gene pool
– may cause cancer
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When and where does genetic
recombination take place?
• During meiosis of human gametes
• In bacteria, occurs when DNA is
transferred between bacteria.
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DNA Recombination Events
•Recombination
- an event in which one bacterium donates DNA to
another bacterium called a recombinant
-
end result is a new strain different from both
the donor and the original recipients
-
depends on the fact that bacteria have
plasmids and are adept at interchanging
genes
-
provide genes for resistance to drugs and
metabolic poisons, new nutritional and
metabolic capabilities, and increased
virulence and adaptation to the environment
Vertical vs. Horizontal Gene Transfer
• Vertical gene transfer=
• Genes/DNA passed from an organism to its
offspring
• Horizontal gene transfer=
• Genes/DNA transferred between organisms
• Which type do humans have?
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Types of Genetic Transfer in Bacteria
Conjugation
transfer of plasmid via sex pilus~conjugal visit b/t bacteria
plasmid=“mini-chromosome carrying extra genes”circular and self-replicating
Transformation
genes transferred from one bacterium to another as
“naked” DNA in solution
Transduction
DNA transferred from donor to recipient cell inside a virus
that infects bacteria(Bacteriophage/phage)
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Plasmids
• self-replicating, gene containing circular pieces
of DNA
• 1-5% the size of bacterial chromosome
• “mini-chromosome”
• Bacteria can store up many different plasmids
for their use & can transfer these to other
bacteria.
– I.e. antibiotic resistance genes, toxin production, etc.
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Antibiotic Resistance (R) Plasmid
• Some plasmids can carry many antibiotic
resistance genes.
• When bacteria collect many plasmids (they can
possess more than one) and these plasmids
have many antibiotic resistance genes, a
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“superbug” may originate.
Mechanism of Conjugation
• A donor cell (called the F+ cell in this
case) contains a F (fertility) plasmid.
• A conjugation pilus forms and the donor
cell transfers a copy of the F plasmid to
the recipient.
• Now, both cells have a F plasmid
• What would happen if the F plasmid was
really an R (antibiotic resistance)
plasmid?
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Conjugation
• Transfer of plasmid DNA from a F+ (F factor)
cell to a F- cell
• An F+ bacterium possesses a pilus
• Pilus attaches to the recipient cell and creates
pore for the transfer DNA
F Factor Transfer
Transfer of the F factor, or conjugative plasmid
Chromosomes
F factor (plasmid)
Donor F+
Bridge made
with pilus
F factor
being copied
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Recipient F–
Conjugation
• High frequency recombination (Hfr)
donors contain the F factor in the
chromosome
Donor
Hfr cell
Partial copy
of donor
chromosome
Integration of
F factor into
chromosome
Bridge
broken
Pilus
Donated
genes
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Transformation
• “Naked” DNA fragments of one disintegrating cell are close to another live
cell.
• Some cells have the ability to “pick up” naked DNA fragments and “insert”
or recombine the DNA into their own DNA
• The new recombinant cell now has its original DNA, plus some new DNA
from the disintegrating cell.
• What if genes a or b were genes for penicillinase (an antibiotic resistance
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gene)?
Transformation
• Nonspecific acceptance of free DNA by
the cell (ex. DNA fragments, plasmids)
• DNA can be inserted into the
chromosome
• Competent cells readily accept DNA,
luckily not all bacteria can become
competent just like not all bacteria form
spores or flagella, etc.
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DNA released from a killed cell can be accepted by a live
competent cell, expressing a new phenotype.
Bacterial transformation
Fig. 9.25 Griffith’s classic experiment in transformation
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Mechanism of Transduction
• When you think of Transduction, think virus
mediated gene transfer
• The virus is able to kill the initial bacterial
cell.
• When the cell lyses, the viral particles which
have picked up DNA from the original cell
now insert that DNA into a new cell.
• The new cell may or may not insert the new
DNA sequence into its chromosome.
• Transduction can be a problem when the
red DNA codes for an antibiotic resistance
gene.
• Can you see how antibiotic resistance can
be transferred?
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Transposons
• Transposons=
• small segments of DNA that can move (be
transposed) from one region of a DNA molecule to
another.
• “jumping genes”
• not a “mini-chromosome”, just a linear segment of
DNA that can jump within one chromosome/plasmid
or between them.
– Involved in
• changes in traits such as colony morphology,
pigmentation, and antigenic characteristics
• replacement of damaged DNA
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• intermicrobial transfer of drug resistance (in bacteria)
Transposons
• Some genes can “jump” from
chromosome to plasmid, from plasmid
to plasmid or from plasmid to
chromosome,
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Genes & Evolution
• Genes are continually altered due to
mutation, recombination, and transposition
• These changes increase genetic diversity of
the gene pool and then natural selection acts
on diverse populations to ensure survival in
many different habitats.
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