Download Chapter 6 Microbial Genetics

Microbial Genetics
• Dr. Gary Andersen, 913-279-2211
• Some slides used with permission from Curtis Smith, KCKCC
• Reference: Chapter 7,8 from (Black, J., 2005)
Basic Units of Genetics
• Genomes – the total of the genetic material
in a cell.
• Gene - The unit of heredity for a given
genetic trait. The site on a DNA molecule
that carries the code for a certain cell
function.
• Viruses – 4 or 5 genes, E. coli – 4228 genes,
Human ~ 31,000 genes.
A Big Question to Struggle With
• Which is more important… nature or nurture?
Genetics or the environment? In
determining the characteristics and behavior of
an organism?
Nucleic Acids
I. Nucleic acids are located in the nucleoid
of bacteria, and the nucleus of
eukaryotes. There are 2 kinds of nucleic
acids: RNA & DNA.
Ruptured
E. coli cell
showing DNA
DNA
A. CHARACTERISTICS OF DNA
DNA (deoxyribonucleic acid) is made of
subunits called nucleotides. Nucleotides are
made of 3 components. These 3 components
are linked together with a covalent bond.
E. Coli = 4.6 million nucleotide pairs (~1mm)
Corn = 2.5 billion nucleotide pairs
Human = 3 billion nucleotide pairs (2nm wide by
2 meters long)
Significance of DNA Structure
• Maintains the code with high degree of
fidelity. (double strand assures accurate
replication)
• Provides a method for introducing a high
degree of variety. (unlimited variety of
sequences possible)
1. COMPONENT 1 - Phosphate
Phosphate group - Phosphate functions as a
structural part of nucleic acids.
2. COMPONENT – Ribose Sugar
2, DEOXYRIBONUCLEIC
ACID
Ribose - A five carbon sugar
that functions as part of the
DNA backbone (ie. structural).
“2, Deoxy” means without
oxygen on the number 2
carbon atom.
3. COMPONENT – Nitrogen Bases
NITROGEN CONTAINING BASES
Function: express genetic information.
composition :
2 PURINES:
ADENINE (A) GUANINE (G)
double ring structures
2 PYRIMIDINES: THYMINE(T) CYTOSINE(C)
single ring structures
Nucleotide Base
Composed of one Nitrogen base, one Deoxyribose,
and one Phosphate group
Deoxyribose
Phosphate
Adenine
(Nitrogen base)
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4 Nucleotides
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DNA Structure
4. DNA is a double helix (there are 2 strands of
DNA) which are intertwined with 5 base pairs
per turn.
5. DNA has complimentarity
that is
A always bonds with T
and
G always with C
6. DNA is always antiparallel. The 2 strands of
DNA are always oriented in opposite directions.
( 5’ PO3 end – 3’ OH end)
http://www.umass.edu/microbio/chime/dna/dna53.htm
DNA Bonds
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3-D Image of
DNA
B. RNA
RIBONUCLEIC ACID
Similar to DNA except:
1. RNA is single stranded
2. RNA has a ribose sugar instead of
deoxyribose. (Oxygen on #2 C).
3. RNA has URACIL (u) instead of thymine
4. RNA is always shorter than DNA, ~ 1,000
nucleotides in length
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C. FUNCTIONS OF RNA
1. rRNA (ribosomal) - comprises the ribosome
(site of protein synthesis). (60% of a ribosome
is made of RNA, the rest is protein).
2. tRNA (transfer) carries amino acids to the
ribosome during protein synthesis. Also
known as the “ANTICODON”
3. mRNA (messenger) - a complimentary strand
of RNA equal in size to 1 gene (normally ~1,000
nucleotides). “CODON” - coded info from
DNA (bound for the ribosome)
THE CENTRAL DOGMA OF
BIOLOGY “Francis Crick – 1956”
There are 3 parts to the flow of
information in all cells.
Transcription
Translation
DNA -------------mRNA-----protein
Replication
Central Dogma of Biology
DNA REPLICATION
1. Where 2 parental strands of DNA are
copied into 2 daughter strands. Rate =
1,000 nucs per seconds without error.
This leads to binary fission in bacteria.
Cell Division) = 2 daughter cells
2. Each cell receives 1 parental strand and
1 daughter strand. (semiconservative
replication)
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As the two replication forks meet, the two new chromosomes
separate—each containing one new and one old strand
Replication bonding
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1. EVENTS IN DNA
REPLICATION
a. DNA unwinds using the enzyme DNA
Helicase
b. SSBP holds the 2 strands apart (single
strand binding proteins)
c. Note: 2 replication forks. DNA
replication is considered bi-directional
replication.
DNA REPLICTION
CONTINUED
d. Polymerization requires DNA Polymerase
(POL III) which is an enzyme that synthesizes
2 nucleotide strands (daughter strands) from
2 parental (templates) strands.
e. DNA exonuclease (POL I) removes any
mistaken base pairs.
f. DNA ligase seals any gaps and joins the 2
strands together.
DNA Replication Enzymes at Work
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Steps in Replication
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Replication of DNA cont’d
• http://www.ncc.gmu.edu/dna/repanim.htm
THE CENTRAL DOGMA OF
BIOLOGY
There are 3 parts to the flow of
information in all cells.
Transcription
Translation
DNA -------------mRNA-----protein
Replication
B. TRANSCRIPTION
1. 2nd part of the central dogma of biology
2. 1st step in gene expression (i.e.protein
synthesis).
3. The cells genetic plan contained in DNA is
transcribed into a complimentary base
sequence called messenger RNA (mRNA).
4. The region of DNA that produces or serves as
a template for mRNA is called a gene. A gene
normally consists of around 1,000 base pairs.
It is the smallest segment of DNA that codes
for mRNA.
TRANSCRIPTION CONTINUED
5. RNA polymerase is the enzyme
responsible for making mRNA
Transcription continued
7. Example:
DNA
DNA
mRNA
A T G C C G
T A C G G C
A UG C C G
8. mRNA is a blueprint of DNA or a
transcript or code.
9. One code word consists of three letters.
Animation of Transcription
• http://www.ncc.gmu.edu/dna/mRNAanim.htm
C. TRANSLATION
1. Translation is the 3rd part of the central dogma of
biology (2nd step in gene expression or protein
synthesis).
2. After transcription, the coded information in mRNA
is translated into an enzyme (protein).
3. This process takes place on the ribosome. Note that
the ribosome is made of rRNA and protein.
Translation Graphic
TRANSLATION CONTINUED
4. tRNA STRUCTURE
tRNA utilizes the information in mRNA
to determine the sequence of amino
acids in a protein. tRNA has a
cloverleaf shape. The amino acid end
binds one specific amino acid in the
cytoplasm. The anticodon end pairs
with the codon on mRNA.
Transfer RNA Structure
TRANSLATION CONTINUED
3. The mechanics of translation
Initiation; mRNA bumps into the small
subunit and triggers the two ribosomal
subunits to bind together. The first
tRNA anticodon (UAC) carrying the
amino acid methionine hydrogen bonds
with the codon AUG on mRNA.
TRANSLATION
CONTINUED
b. Elongation – The second tRNA
binds to the second code word on
mRNA. A peptide bond forms
between the two amino acids. The
first tRNA leaves, and the enzyme
translocase moves the ribosome
down one code word of mRNA at a
time. This repeats ~ 300X.
TRANSLATION
CONTINUED
In termination, one of three
possible stop codons is reached.
The last tRNA falls away and the
two ribosomal subunits fall apart.
C.
d. The Genetic Code
61 sense codons for 20 amino
acids
3 nonsense (or stop codons)
64total codons
Pg 180 (Black, J., 2005)
The Genetic Code
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Steps in Protein Synthesis
Steps in Protein Synthesis
Steps in Protein Synthesis
Steps in Protein Synthesis
Steps in Protein Synthesis
Protein Synthesis
Translation - Animation
• http://www.ncc.gmu.edu/dna/ANIMPROT.htm
Translation Animation - http://www.wehi.edu.au/wehitv/dna/movies/Translation.mov.gz
e. Translation Blockers
1. Streptomycin – (SM) blocks assembly of
the ribosome during initiation.
2. Chloroamphenicol – (CA) blocks peptide
bond formation during elongation.
3. Tetracycline – TC – blocks the 2nd site on
the ribosome during elongation.
4. Erythromycin EM – blocks translocase
during elongation.
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Gene Regulation
• How can genes be turned off and on?
• Examples from E. coli
– Inducer – example is lactose (lac operon), pg
187 of (Black, J., 2005)
– Repressor – argenine (arg operon), pg 187-188
of (Black, J., 2005)
Induction - Lac operon
Repression - Trp operon
III. 5 Ways of Creating
Genetic Diversity in Bacteria
A.
B.
C.
D.
E.
Mutations
Transformation
Conjugation
Transposition
Transduction
A. Mutations
1. Changes in the nucleotide sequence
usually due to an error in DNA
replication. These occur naturally at
low levels (also known as spontaneous
mutations); or by the effects of
chemical agents called mutagens; or
by physical agents like radiation.
Results of Mutations
2. Most mutations are neutral - they have
no effect on the polypeptide. Some
mutations result in a less active product;
Less often an inactive product; Very few
mutations are beneficial. However, these
would be passed on!
Types of Mutations
3. Point mutations - a one base change in
DNA. There are 3 types:
a. silent mutations - single base
substitution in the 3rd base nucleotide
position of a codon. This results in NO
change in amino acid. Note that the
first 2 letters of the genetic code are the
most critical.
b. missense mutations - single base
substitution in 1st or 2nd base nucleotide
position. This results in a changed amino
acid. A change in one amino acid usually
will have little effect depending on where in
the polypeptide it occurs.
c. nonsense mutations - single base
substitutions that yield a stop codon. Note:
there are 3 nonsense codons in the genetic
code = NO PROTEIN
4. Frame Shift Mutations - the addition or
deletion of 1 or more bases. These are
due to powerful mutagens; chemical or
physical.
a. Chemical mutagens - (used in research to
study mutagenesis). There are 3 kinds of chemical
mutagens.
1. alkylating agents. Adds alkyl group, CnH(2n+1) Ex.
formalin, nitrogen, mustard, and ethylene oxide
(reacts with G changing it to bind with T).
2. base analogs. Mimics a nitrogen base. Ex. AZT is
a modified sugar that substitutes for T. Ex. 5 bromouracil binds with A or G.
3. intercalating agents. Inserts into DNA and
pushes bases apart. Ex. AFLATOXIN - a chemical
produced by peanut and grain molds. The mold is
Aspergillus flavus (fungus).
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b. Physical mutagens:
1. nonionizing radiation - Causes the
formation of T= T dimers. UV light
@ 260 nm.
2. Ionizing radiation - damages DNA
by causing the formation of “free
radicals” leading to mutations. 3 Ex.
X-rays. Gamma rays from
radioactive fallout penetrates the
body. Alpha rays from inhaled dust
containing radioactive fallout.
B. TRANSFORMATION
The passage of homologous DNA from a dead
donor cell to a living recipient cell. Occurs in
Streptococcus pneumoniae. When S. pneumo
dies the DNA can be absorbed by a living S.
pneumo and recombined into the chromosome.
The gene for capsule formation is obtained in
this way, as is a gene for penicillin resistance.
Discovered in 1929 by Fredrick Griffith.
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Griffith’s Transformation Experiment
C. CONJUGATION
1. A “mating” process between a donor
F+ (bacteria with fertility factor =plasmid)
and an F- recipient cell.
2. Occurs in Gram - enteric bacteria like
E.coli
3. Discovered in 1946 by Joshua Lederberg
and Edward Tatum.
4. Plasmids carry genes that are nonessential
for the life of bacteria. Ex. gene for pili (sex
pilus). Ex. plasmid replication enzymes.
Ex. Medical Problem: R-Factor = antibiotic
resistance!
Conjugation continued
“Normal - Sex” plasmid transfer
(usually ~20 of 100 genes).
a. Requires a sex pilus
b. F + bacteria transmits a copy of the
plasmid
to F- bacteria. This converts the F- cell into an F +
cell. Medical Problem: The R factor (antibiotic
resistance) on the F factor is transmitted!
http://www.cat.cc.md.us/courses/bio141/lecguide/uni
t4/genetics/recombination/conjugation/f.html
6. Hfr (High Frequency Recombination)
a. Hfr- bacterial plasmid integrates into the
chromosome.
b. Medical Problem: Hfr antibiotic resistance
genes are passed during binary fission
(every time the cell divides). Therefore,
antibiotic resistance spreads very rapidly!
c. When Hfr mate with F – bacteria, only the
bacterial genes cross NOT plasmid genes.
Genetic diversity results in this case due to
recombination.
http://www.cat.cc.md.us/courses/bio141/lecguid
e/unit4/genetics/recombination/conjugation/hfr.
html
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D. TRANSPOSITION p 285
1. Transposons (jumping genes) are big
chunks of DNA that randomly excise
and relocate on the chromosome.
2. Transposons were discovered in 1950
by Barbara McLintock in corn.
3. Causes antibiotic resistance in Staph.
aureus, the famous methicillin
resistant Staphlococcus aureus (MRSA)
strain!
E. TRANSDUCTION
the transfer of genetic material from
donor bacteria to recipient bacteria via a
transducing agent (virus!). Bacterial
viruses are called bacteriophage.
1. Discovered in 1952 by Zinder &
Lederberg.
2. Two kinds of transduction:
generalized and specialized.
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2. Generalized transduction: Starts with the
LYTIC CYCLE where a T- even phage (Fig.
8.5 pg 210) infects E.coli killing the host cell,
and synthesizing 2,000 copies of itself. The
T-even phage randomly packages bacterial
DNA in a few defective phages. Once a T –
even phage infects another E. coli, this
genetic information can be recombined into
the host cell without causing the lytic cycle.
New genetic information is thereby
transduced from one bacteria to another.
Generalized Transduction
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Generalized
Transduction
Specialized Transduction
3. Specialized transduction
Lambda phage infects E.coli. The phage does not
lyse the cell immediately. Instead it integrates
into chromosome of the bacteria as a prophage
and remains dormant. This is called the
LYSOGENIC CYCLE.
Phage genes are
replicated and passed to all daughter cells until
the bacteria is under environmental stress,
from lack of nutrients, etc. Then phage gene
will excise from the nucleoid and enter the
LYTIC CYLE taking one adjacent gene for
galactose metabolism.
Specialized Transduction Cont.
The gal transducing phage (lambda)
makes ~ 2,000 copies of itself with the gal
gene, and infects other E.coli. When gal
integrates into the nucleoid of other E.
coli, it may provide these bacteria with a
new capacity to metabolize galactose.
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Comparison of Bacteriophage
3. Comparison of bacteriophage
transduction in E.coli.
Generalized
Specialized
T even phage
lambda phage
lytic cycle
lysogenic
random packaging
specific gal gene
End of Slides