Download Bacterial Genetics

CLASS: 11-12
DATE: 10-18-2010
PROFESSOR: Adrie JC Steyn
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BACTERIAL GENETICS
Scribe: Christine Sirna
Proof: Louisa Warren
Page 1 of 9
BACTERIAL GENETICS [S1]
a. Information is basic. However, I can come up with some trivial questions that few people can answer
b. There are fundamental principles there that you should know
c. I will point out important areas and areas that are not important and that you shouldn’t know
COURSE GOAL [S2]
a. READ SLIDE
b. Mechanism is everything! There is no use observing things. The key thing is to figure out how things work.
CONTENT [S3]
a. To start off I will talk about DNA replication but more specifically DNA the genetic material
b. How does DNA replicate in bacterial cell
i. For example you have a bacterial cell that divides and when cell divides, DNA has to duplicate or replicate,
exact copy needs to replicate with the organism or the organism wouldn’t survive.
c. Transcriptional control.
i. Do you know what it is? The Current Dogma
1. You have and DNA is transcribed to transcripts which get translated into proteins
d. Mutation Repair and recombination
e. Gene exchange and genetic transfer and lastly genetic engineering
i. Gene exchange and genetic transfer are very important,
1. Antibiotic resistance. How does it arrive? How does it occur? what are the mechanisms involved?
2. How does genetic material get transferred from one organism to another.
f. How does drug resistance occur
DNA: THE GENETIC MATERIAL[S4]
a. To start off with this is a typical bacterial cell.
b. Have cell wall, periplasmic space, peptidoglycan, inner membrane, cytoplasm with chromosomal DNA
i. Some bacteria contain plasmid DNA
c. Here is an example of a plasmid DNA
d. Most bacteria have 1 circular DNA chromosome ranging from 1 megabase to 8 megabase or 8,000kb
i. If talk about bacterial chromosome we refer to collection of all genes present on a bacterious chromosome
or its extrachromosomal genetic elements
e. We talk about the genome we talk about chromosome as well as plasmid. We talk about all the genes in the
organism whether they are on the chromosome or the plasmid. It doesn’t matter
f. Plasmid is an extra chromosomal genetic element
i. Replicates independently from the chromosome
ii. Usually small but can be big too
iii. Hydrobacterium has a huge plasmid
iv. E Coli for example has very small plasmid
v. We can easily isolate or separate these plasmids from DNA from chromosomal DNA
1. You can isolate both
2. Through genetic engineering and gene cloning you can transform it etc etc
g. Importantly the genomes contain operons. Which we will discuss later
h. Operons are genes that are arranged in a specific manner
i. For example you have 3,4,5,6,7, genes next to each other, very closely, and sometimes they overlap with a
couple of bases and they are in control of a single promoter
i. Operons are made up of genes (sometimes up to 10-12 genes) in a string with one promoter that controls all
these genes like a light switch. Flip switch on and all genes turn on and they are all transcribed and
translated
j. The promoters and operators control these genes
i. Think of the promoter as the light switch
ii. Genes are switched on or off in terms of transcription
iii. Genes are being transcribed and once the transcripts are formed the genes are being translated into active
proteins
iv. This is an example here of when the genome of an organism has been sequenced and they present these
maps like that is 4.4 million bases and nowadays they do this very quickly. Way back it two years now it
takes 4 hours to sequence the whole genome of an organism.
NUCLEIC ACID… WHERE IS IT? [S5]
a. Nucleic acid where is it? It’s everywhere. All living things contain DNA or RNA
b. DNA or RAN can exist in single or double strand
c. All living things contain nucleic acid which includes DNA or RNA
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 2 of 9
d. When you talk about bacteria that DNA or RNA can be isolated for example here is a gel agarose where we
separate for example isolate the plasmid DNA. Here is a molecular standard here is a plasmid DNA that has
been isolated and again it is an agarose gel
i. Have the Agarose gel and you stain with ethidium bromide which is a carcinogen
ii. Ethidium bromide intercalates with DNA and put in on UV light blocks and DNA fluoresces very clearly.
That is how we do it in the lab to isolate the DNA for PCR
e. Point is all living things contain DNA or RNA whether single of double stranded doesn’t matter
VI. DNA STRUCTURE [S6]
a. DNA structure contains 2 strands which are held together by H bonds
i. Have 4 bases: adenince, guanine, thymine, cytosine
b. Adenine binds to thymine and contains two H bonds
c. Guanine binds to cytosine and contains 3 H bonds
d. G-C bonds are much stronger and harder to break them but they can be broken because they have 3 H
bonds whereas A and T only have 2 bonds
e. Since this is a double helix DNA can be denatured or if you heat it up DNA you can separate these 2 strands
by breaking the H bonds. At about 90 degrees Celcius the two strands get separated.
f. If for example DNA is GC rich it is very difficult to separate the DNA strands. If it is AT rich is separates very
easily through heat.
i. Microbacterium tuberculosis is a high GC rich organism and the strands are difficult to separate
g. And It can reanneal. You can separate both strands of DNA and you can independently add them together
and slowly cool temperature and the two strands will reanneal again
VII. PICTURE OF DNA[S7]
a. SKIPPED
VIII. DNA REPLICATION [S8]
a. When bacteria divide the DNA is duplicated
b. For example here this is a simplistic model of how bacteria like E Coli divide
c. Again you have a complex chromosome that is about 4-8 million bases all intertwined with DNA strands
d. That chromosomal DNA must be precisely duplicated as the organism replicates
e. For example here you have the cell wall and the plasma membrane and the replicated DNA molecules here.
i. The cell wall of plasma membrane begins to invaginate and forms a cross wall, divides the DNA and the
cell separates
f. There are thousands of proteins involved in a typical replication event
g. For example the chromosomal DNA has a specific locus, a specific region, where DNA replication starts and
this is termed the Rec region. Replication initiated at Rec.
i. It requires many enzymes for example helicase
ii. You have the Primase
1. DNA has to be nicked and that nicked DNA is recognized by primase with a specific protein. The 5’
phosphate or 3’ hydroxyl group are recognized by specific enzymes and used as a template for DNA
replication.
h. The new DNA is synthesized Semiconservatively and proceeds bidirectionally
IX. PICTURE OF DNA REPLICATION [S9]
a. Two strands have to be unwound by helicase
b. There are thousands of proteins involved in this process each with a precise function to allow precise
duplication of the chromosomal DNA
c. If it’s not precise there are errors and mutations and the cell most likely will die
d. In most bacteria chromosome is circular not linear
i. Here you have enlargement of the replication fork
ii. Here replication occurs bidirectionally in both directions
iii. Thousands or proteins participate in this process
e. Here we have the replication fork
f. Need to know semiconservative replication!
i. One strand is from the parent and the other strand is newly synthesized semiconservatively
ii. Again this occurs bidirectionally and you get an exact replica of the bacterial chromosome
g. In other words the following separation, the final separation, two chromosome exist and each chromosome
contains one old and one new strand of DNA
h. This is extremely complicated and in most cases we don’t know exactly how this happens
i. The important term is semiconservative and bidirectional
X. DNA DUPLICATION IN THE LAB- THE POLYMERASE CHAIN REACTION [S10]
a. How do we duplicate DNA in the lab? Do you know PCR? Do you know the principles of PCR?
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 3 of 9
b. PCR is Polymerase chain reaction and in simple terms it is a way of amplifying minute quantities of DNA or
RNA. You will have a very small amount of nucleic acid for example DNA actually from a single bacterial cell
that has been done.
i. You can dilute bacterial cells up to the point where you have in a tube one bacterial cell
ii. And you can amplify milligram quantities of that DNA
iii. You can cut a mm of hair and put in a test tube and amplify it and get massive quantities of DNA
c. PCR helps us to amplify
i. Many years ago you had to isolate chromosomal DNA or plasmid DNA from bacteria and you would cut it
with restriction enzymes to perform cloning experiments and you would size select DNA separate by
agarose gels and you would use it in your experiments
ii. Nowadays you take a bacterial colony and put it in this reaction and amplify the DNA on the chromosome
you want
iii. You can amplify any DNA on a bacterial chromosome if you know the sequence of the DNA and this is the
principle
d. DNA duplication in the lab the PCR reaction for example
i. Here you have your DNA strands, imagine you have the whole chromosomal DNA, and you isolate from
the organism and you can take a little of that and again if you know the sequence you can amplify any gene
of your interest
e. You can make use of oligo nucleotide primers specific for your target DNA fragment
f. GOES TO BOARD TO DRAW
i. Ok so this is your DNA
ii. We know the sequence of DNA (AGC or T) and now for example let’s illustrate it this way (draws linear
DNA)
iii. This is your DNA and here you have a gene
iv. You want to amplify this gene
1. The first thing you do is to synthesize two oligonucleotides specific for target DNA
you synthesize for example an oligonucleotides of this sequence (one linear DNA) about 20 bases
and you synthesize an oligonucleotide that is identical to this sequence (the other linear DNA) about
20 bases
2. In other words if you separate these two strands this oligonucleotide will anneal one of the strands
and the other oligonucleotide would anneal to the other strand
g. Annealing is very fast for example a while ago I talked about denaturation and heat can denature the two
strands
i. When you increase heat the two strands separate
ii. If you take these two oligonucleotides and you add it to the separated DNA and you cool it down slowly
these two nucleotides would anneal to the complementary DNA
iii. You separate the DNA in the presence on heat and as you cool it the oligonucleotides within seconds will
anneal to the now separated DNA. This is very fast and very effective
iv. You denature the DNA at 95 degrees and the double stranded DNA now becomes single stranded
v. Here you have the oligonucleotides that are specific for a gene.
h. This oligonucleotides as you decrease the temperature to about 40-65 degrees the oligonucleotides now
anneal to the single strand DNA
i. So now (he makes a double strand then denatures it to make two single strands) the one oligo will anneal to
one strand the other oligo will anneal to the other strand. Very rapidly very quickly
j. This is important that we understand the principles of PCR!
k. Once the oligo are annealed a protein, for example Tac polymerase, is also present in test tube. Tac
polymerase are isolated from Thermus Aquaticus one of these bacteria that grows underseas near the
volcanoes and grows at about 92 degrees celcius
i. The enzyme is very heat stable and this enzyme recognizes the region in front of oligo and starts to extend.
ii. Now you have double stranded DNA. So from one DNA now you have two DNA molecules
iii. In the next round exactly the same is going to happen
l. Now when you denature you have two strands from one molecule and two strands from the other molecule
when you denature
i. So you have one 1,2,4 and the same will happen again
ii. The oligo will anneal and the enzyme will extend the reaction to get double strand DNA. This increases
logarithmically. You get 2,4,8,16,32, 64,128, 256
iii. In other words within 20-30 cycles you get a massive increase in DNA until reagents run out
1. Oligos get used up initially you add in excess but then the free oligos get less and less and less
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 4 of 9
2. Enzyme gets hit by high temps and run out of dNTPs because in this reaction mixture you also have
dNTPs. In other words you have adenine, guanine, cytosine, and thymine and they get used up as
the DNA increases because they get put into the DNA
m. Important point is you have one molecule and 1 becomes 2 which becomes 4 etc
i. In other words it is a repetitive cycle
1. The oligos anneal and in the second strand are being synthesized by Tac polymerase the enzyme
extends the single strand which becomes double stranded and then it goes through the cycle.
2. That is why this machine is called a thermo cycler
a. It increases the temperature and denatures the DNA, the oligos anneal and you slightly
decrease the temperature to 72 degrees and then the enzyme extends it and makes a double
strand for about two minutes.
b. Then it increases the temperature again at 95 it denatures the DNA and separates the two
strands the temperature goes down again and the oligos anneal and the enzymes extends it
and so goes the cycle on
XI. TRANSCRIPTIONAL CONTROL [S11]
a. Within two hours you can amplify the DNA from small to large quantities
b. Very important to understand this principle!
c. They have these hand held monitors now that you can work in Africa or Asia or remote areas that are battery
powered that work quickly within an hour
i. If you want to screen for pathogens and you know the genetic sequence whether it is the Ebola virus or
whatever you can now take a sample of tears, urine or whatever. You put it in the tube with two known
oligos and with the handheld PCR machine you can amplify the DNA. And if you get a band you know the
pathogen is present in the sample.
1. This is a rough indicator for HIV samples, serum samples and this is done frequently
d. Let’s look at transcriptional control. THIS IS IMPORTANT!
e. READ SLIDE
f. Importantly we are going to look at the two component system in bacteria. This is very important because
it’s the way bacteria sense the environment
i. Don’t just think that bacteria grow.
ii. It grows in response to a particular environment. If it doesn’t like the environment it stops.
1. Some enter a dormant state. They don’t like the environment. It may be too hot, too many nutrients
of a particular kind that inhibit growth.
2. Somehow the extracellular environment no matter where it is, the bacteria has to sense this
environment. It always senses the environment
3. In response to the environment it will induce the expression of very specific genes such as
pathogens
g. When you culture a pathogen on a Petri dish in the lab a certain set of genes will be expressed because it
has been cultured on Lb medium
h. As soon as the pathogen invades a host, such as a human, it doesn’t see LB medium anymore now it sees a
completely different environment.
i. Host cell material, different temperature ions, nutrients
ii. Now a different set of genes are being expressed. Why? Because the environment is different
i. No organisms maintains its overall gene expression levels
i. That is why we study the lac operon
ii. Because you can add a component for example E Coli and a specific gene will be switched on. If you add
lactose the gene will be switched on.
j. You need to make the mental leap from what we do in the lab to pathogens
k. The mechanism is important because once you understand the mechanism, for example how the lac operon
functions you can extrapolate that to many other pathogens in terms of how they respond to a different
environment. That is crucial.
l. If you know how bacteria respond to different environments with a specific mechanism you can design two
things: drugs cause now you know how it works and If you don’t know how it works you cannot develop a
good vaccine.
XII. CENTRAL DOGMA: DNA RNA  PROTEIN [S12]
a. Central dogma is very important
b. All of you should know that once you have DNA this can be transcribed to generate transcripts
i. You have a DNA double helix here after transcription you have a single mRNA molecule
ii. This process is called transcription
c. After the mRNA is generated translation takes place to generate the protein from mRNA
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 5 of 9
d. DNA  transcript protein is very important
XIII. BASIC PROKARYOTIC GENE ORGANIZATION [S13]
a. This is an example of an operon
i. For example here you have a piece of DNA and here you have four genes (ABCD)
ii. Each gene encodes a particular protein
b. This is In an operon because all 4 genes are in the control of the promoter
i. This promoter region controls the expression of ABC and D
ii. This is the start of the operon and this is the end of the operon
c. RNA polymerase binds to promoter region in a particular way and switches the genes on and transcripts are
generated until it reaches termination sequence
i. The termination sequence is a particular sequence that has been recognized by RNA polymerase and tells
the RNA polymerase to stop. This is the end of the gene
d. Then mRNA molecule is generated which is then translated to 4 individual proteins
i. These genes can be next to each other. What does next mean?
ii. Can be 1,2, 5,10,20, or 80 bases or even 100 base pairs away from each other
iii. In other words between these two genes can be between 1 and 80 base pairs
iv. The genes in an operon can overlap also
e. Typically the spaces are not usually more than 100 base pairs. So that is easy to identify
f. The important point here is this is the promoter region.
i. The TSS is the transcriptional start site
ii. Transcriptional start site is where the transcript starts at the +1 position
iii. Now you see that the first gene starts at A and transcript starts before the start of the first gene
iv. So you have have a long transcript here and that is all necessary for effective transcription
g. Because the translational machinery recognized that this is the start of the mRNA molecule but the actual
start of the gene starts here and it recognizes an ATG to translate the mRNA molecule to generate for
example this alpha protein
h. This is the promotor region which consists of -10 to -35 region
i. These are two regions where RNA polymerase binds
ii. It recognizes the -10 and the -35
iii. Why -10? -10 base pairs from the +1 position
iv. In most cases but not all cases have a specific sequence at -10 and -35
i. RNA polymerase is complex molecule with many subunits to which a lot of regulatory proteins bind to
modulate gene transcription. WHY?
i. Because regulatory proteins or transcription factors usually respond to environment signals
ii. And in response to a particular environment a regulatory protein binds to RNA polymerase and says I am
experiencing this environment so switch these genes on
j. From different environments you have different transcription factors or regulatory proteins that bind to RNA
polymerase to tell RNA polymerase to start transcribing or stop transcribing
XIV.
PROMOTER PROPERTIES [S14]
a. Now we zoom in on promoter properties
b. Again This is important
c. Here we have the transcriptional start site where the mRNA molecule starts
d. At ATG position which encodes for MET you have start of a translated protein
e. So the transcriptional start site and translational start site are two different things.
f. Here is the -10 region here is the -35 region.
g. E Coli typically has a TATAAT sequence. This is a conserved sequence but you get some variation. In some
cases you cannot identify the -10.
h. Certain genes are regulated differently so they may not have this consensus sequence but most genes have
this sequence, the -10 and -35 region
i. This is consensus sequence for most genes in for example E Coli
XV. PICTURE OF DNA REPLICATION [S15]
a. It makes the point here that RNA polymerase touches the DNA here and it touches the DNA there and by
touching the DNA, the touching is influenced by different regulatory proteins
b. The function of RNA polymerase
i. RNA polymerase binds to DNA in a specific fashion
ii. For example here we have the promoter sequence and we have the downstream sequence and the
termination. In other words transcription occurs from the left to right direction in this picture until it reaches
the terminator and then it stops.
c. There are 4 steps of transcription or how RNA polymerase initiates transcription
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 6 of 9
i. First RNA polymerase binds to the DNA promoter and unwinds the DNA
1. It binds to this site now you have a double helix must be unwound in the opposite direction. It has to
be opened up
ii. Elongation is the next step
1. This is where the RNA polymerase moves from the 5’ Phosphate to the 3’ hydroxyl group and here
RNA is being synthesized
iii. You have continued elongation where the DNA is being unwound and it moves along with RNA polymerase
and you get this production of a long mRNA molecule until it reaches the terminator site where RNA
polymerase is being released from the DNA molecule. This happens very fast
d. Irrespective of whether the organism grows fast or slow. Replication is distinct from transcription.
e. So you have initiation,, elongation, continued elongation and termination. 4 basic steps that you should know.
XVI.
THE LAC OPERON (NEGATIVE REGULATION) [S16]
a. Let’s look at the lac operon.
b. This is an example of negative regulation in this particular example
c. Here we have a DNA molecule with 3 genes
i. The B galactosidase, permease, and transacetylase
d. Here we have the promoter region and here we have an operator sequence to which RNA polymerase binds
e. Upstream of this operon you have this I gene or repressor gene under the control of its own promoter. Again
an operon is a bunch of genes next to each other under the control of a single promoter.
i. When the repressive protein is being produced and it is being transcribed and translated into this active
repressor. This active repressor can bind to operator sequence, a conserved operator sequence. And when
it binds to this operator sequence, RNA polymerase cannot transcribe these genes. In other words no lac
mRNA is being produced and subsequently no protein is being produced
f. Repressor protein binds to the operator sequence adjacent to the promoter
i. Usually operator sequence overlaps with promoter region
ii. The repressor molecule competes with RNA polymerase at the repressor site. In other words at very low
levels this repressor has no effect
iii. As soon as you increase the repressor levels, you affect the function of RNA polymerase, which results in
no mRNA and no protein. Transcription of the genes is blocked
g. This is an example of negative regulation. In other words, you get the production of a repressor. The
repressor binds to the operator sequence and prevents transcription, prevents translation and you get no protein
XVII. INDUCTION OF THE LAC OPERON [S17]
a. Again here you have the lac operon with three genes.
i. You have the promoter and the operator sequence and here you have the gene that encodes for the active
repressor. Now if you add lactose to the media for example in the case of E Coli.
ii. Lactose is the inducer and binds to the active repressor once it binds to the repressor the repressor can’t
bind to the operator sequence.
iii. If it doesn’t bind to the operator sequence RNA polymerase is fully capable of transcribing these genes
yielding an mRNA molecule that has been translated into 3 different proteins
1. B galatosidase, permease, and transacetylase
b. In other words, as soon as lactose is present the lac genes can be expressed lactose can then be utilized by
the cell
c. The basic function on how the lac operon operates you guys have to know. The concept of an operator, or a
repressor binding to an operator preventing functioning of RNA polymerase, which prevents transcription and
prevents translation. You need to really understand this.
XVIII. POSITIVE CONTROL OF THE LAC OPERON [S18]
a. Exactly as it was before except remember I talked about transcriptional factors or regulatory proteins
i. In different environments, different bacteria senses different environment and that information has to be
relayed to the gene transcription level and it does so via these regulatory proteins or transcription factors
b. These regulatory proteins or transcription factors exert there mode of action by binding to RNA polymerase
i. For example in this case you have CAP the catalytic gene activator protein or another name is the CRP.
ii. CAP binds to RNAP when cAMP is present
c. Now in the presence of poor media with lactose the organisms cannot grow well the following happens
i. in the presence of lactose for example, the repressor is produced and transcribed to produce an active
repressor. You can add lactose which binds to the repressor but now the presence of this protein prevents
binding of this complex to RNAP and eventually you get the synthesis of these 3 proteins
d. This is an example of positive control as opposed to negative control of the lac Z operon because of the CAP
protein
i. It is an accessory protein, a regulatory protein, a transcription factor
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 7 of 9
ii. There is few media, few nutrients available however in the presence of lactose this occurs with the lac
operon. This is an example of positive control.
XIX.
IN SUM… [S19]
a. READ SLIDE
b. For example, CAP is an example of an apo-inducer
XX.
TWO COMPONENT SYSTEMS IN BACTERIA [S20]
a. I’m going to give you a more simplified version then what is on the slide (He goes to draw on the board again)
b. You have a cell membrane (points to the inside and outside of a cell membrane)
i. The cell membrane of an organism in response to a particular environment (temp, pH, nutrients, anything,
osmotic environment, anions, cations)
ii. It can sense that there is a new environment
c. For example, Let’s talk about a lung pathogen, such as microbacterium tuberculosis
i. Once it enters the lung it senses post lipids for example for ions like K. How does it do so?
1. Does so via two component proteins
2. Most bacteria if not all have a two component protein
d. For example you have a protein (typically transmembrane unless it’s called a HIS kinase)
e. Why is it called a HIS kinase?
i. HIC senses a particular molecules that can be O, low O or high O, and it senses it through a particular
sequence on a protein
ii. Once it senses it, it first auto-phosphorylates itself and then it transfers this Phosphate group to a
transcription factor or response regulator (RR)
iii. So it sense a signal via a specific domain that is very complicated
1. 99% of the time we don’t know how this happens
2. Sometimes it asks for steady groups for example a heme group. An O can bind to the heme group or
NO can bind to the heme group
f. But how does it sense pH or temperature?
i. Most cases we don’t know
ii. And because it senses it, once it’s bound to this molecule there is a conformational change, it
autophosphorylates itself and again once it’s phosphorylated it affects the histidine kindase activity and it
transfers this phosphate group to its corresponding partner the response regulator.
iii. That is why it’s called a two component system
iv. Usually it’s two proteins in operon next to each other
1. One senses the environmental signal and via autophosphorylation and dephosphorylation, it
transfers P to it’s corresponding partner the response regulator
g. This RR induces gene expression via binding to RNA polymerase
h. This is illustrated here. Here we have membrane protein for example the KdpD and KdpE component system.
It’s two proteins and an operon that can respond to any of these signals
i. It has a sensor domain that sense the signal and via phosphorylation, autophosphorylation, and then
dephosphorylates itself, it transfers the phosphate group to this protein KdpE
1. KdpE binds to RNA polymerase which consists of these subunits to activate gene expression
i. Bacteria have many of thee two component systems
j. Again it’s two components because one senses the signal and the second one transmits the signal in terms
of gene expression
k. That slide is important
XXI.
MUTATION, REPAIR, AND RECOMBINATION [S21]
a. Very simple
b. If you can read you can understand it
XXII. GENERAL [S22]
a. Read for yourself
XXIII. SINGLE POINT MUTATIONS [S23]
a. An example of transition is a purine replaced by a purine or a pyrimidine replaced by a pyrimidine
b. An example of transversion is a purine replaced by a pyrimidine or vice versa
XXIV. PICTURE OF THYMINE DIMERS [S24]
a. Exposure of DNA to UV light results in formation of thymine dimers, generates a mutation
b. For example, skin cancer
c. And this cannot act as a template for DNA polymerase
XXV. DNA REPAIR MECHANISMS [S25]
a. Another example of mutations
b. There are a couple of DNA repair mechanisms
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 8 of 9
c. This is simple you guys can read through this
d. If I say this is simple, rip the pages out because I might make a question or two out of it
XXVI. GENERAL EXCISION REPAIR [S26]
a. Here you have a damaged base that has been recognized by an endonuclease
b. An endonuclease is an enzyme that typically recognized DNA changes in the middle of the DNA
c. Exonuclease is at the sides of DNA molecules
d. Exonuclease carries out excision of DNA fragment where DNA polymerase pulls it in and DNA ligase seals
the nick
XXVII. SILENT MUTATIONS [S27]
a. It’s simple you guys can go through that
XXVIII. GENE EXCHANGE IN PROKARYOTIC CELLS [S28]
a. Familiar concepts: transformation, transduction, and conjugation
b. Transformation: for example the uptake of naked DNA for example some bacteria release DNA and other
bacteria just take that released DNA up
c. Transduction is bacteriophage mediated DNA transfer. Some bacteria is infected with a bacteriophage with a
phage it has inserted itself into the DNA. Then the phage inside the bacterial cell starts replicating until it
reaches a point where the bacterial cell lyse and it releases millions of phages which can reinfect other
bacteria. As it does so, it grabs some other bacterial DNA and takes it with it and transfers this DNA to other
bacteria.
d. Conjugation: DNA can be transferred via conjugation
XXIX. TRANSFORMATION AND TRANSDUCTION [S29]
a. An example of tranformation where the DNA is being released and taken up
b. AN example of transduction, here you have the bacteriophages that gets released from bacteria that lyse the
bacteria and reinfect other bacteria.
XXX. CONJUGATION [S30]
a. You have the conjugation tube where nucleic acid or DNA can be transferred from one bacterial cell to
another bacterial cell. These are very simple concepts
XXXI. PLASMIDS CONTAIN GENETIC INFORMATION THAT CAN BE EXCHANGED [S31]
a. This is an example of a plasmid that can be genetically engineered
XXXII. BACTERIOPHAGES [S32]
a. Again a bacteriophage recognizes a receptor on the bacterial cell, injects its nucleic acid that integrates into
the chromosomal DNA and the bacterial cell starts synthesizing bacteriophages.
XXXIII. LYTIC LIFE CYCLE [S33]
a. For example in the lytic life cycle, the bacteriophage attaches itself to the bacterial cell, injects the DNA, then
DNA integrates into the chromosomal DNA of the bacterium. This chromosomal DNA, this newly inserted
DNA now encodes for phage particles.
b. Then the phage particles accumulate and up to a certain point the bacterial cell gets lysed and releases
thousands of bacteriophages, which reinfect new bacteria.
c. The lysogenic life cycle refers to when phage DNA replicates with the bacterial chromosome. In other words,
lysing does not occur. The phage inserts itself into the cell, integrates into the DNA and replicates with the
bacterial cell. Does not cause lysis.
XXXIV. GENERALIZED TRANSDUCTION [S34]
a. SKIP
XXXV. TRANSPOSON [S35]
a. An example of mobile genetic elements that can transfer DNA within itself from one position to another in the
genome or between different molecules of DNA.
i. From plasmid to plasmid or plasmid to chromosome
b. It’s a weird piece of DNA that can jump around randomly
i. It encodes for own proteins
ii. This allows it to be excised from DNA and it jumps from to a different region in the chromosome or it jumps
to a plasmid. Just think about it. If it jumps randomly anywhere and anyplace you can mutate certain genes
1. The mutated gene cannot encode for active protein
2. But it has all the sequences: recombinases, resolvases to jump around
c. Most transposons have antibody markers
i. Encodes for drugs, for resistance to certain drugs
d. What happens is one pathogen can get lysed, release all the DNA containing a transposon. It can be taken
up by a different pathogen, this naked DNA. Now this different pathogen can become drug resistant and can
transfer DNA through transformation, conjugation, and transduction
e. There is about 100 million bacteriophages in sea water
CLASS: 11-12
Scribe: Christine Sirna
DATE: 10-18-2010
Proof: Louisa Warren
PROFESSOR: Adrie JC Steyn
BACTERIAL GENETICS
Page 9 of 9
f. DNA is constantly being exchanged between different populations
XXXVI. TRANSPOSITION [S36]
a. These are the sequences. You have a transposon and it jumps. Gene B is now disrupted
XXXVII. Tn3 MEMBER OF THE TnA TRANSPOSON FAMILY [S37]
a. This is for example the genes on the transposon
b. You have this transposase, resolvase, and the antibody marker
XXXVIII.
CHROMOSOMAL DNA CAN BE EXCHANGED [S38]
a. Here you have an organism that contains a chromosome and the organism contains a capsule
b. This organism can get lysed release the DNA and now it can be taken up by a different bacterium that is not
capsulated
c. Because the genetic material has been transferred from a capsulated organism to a non capsulated
organism, this non capsulated organism can now become capsulated
d. The same is true for antibiotic resistance
i. This is where you get outbreaks in hospitals because these Antibody markers are transferred through
transformation, transduction, and conjugation
XXXIX. IMPORTANT- PLASMIDS CONTRIBUTE TO THE EVOLUTION OF ANTIBIOTIC RESISTANT [S39]
a. This is a diagram to illustrate basically it summarizes all 3 events
b. You guys can go through this
c. Important slide
XL. GENETIC ENGINEERING [S40]
a. skip
XLI.
PICTURE [S41]
a. skip
XLII.
RESTRICTION ENZYMES [S42]
a. This is an important slide
b. DNA. Some wonder how do you clone?
c. We know we can amplify DNA
d. IF we want to clone DNA we can make use of restriction enzymes and there are a couple thousand of theses
restriction enzymes and they recognize DNA
i. They recognize specific DNA and we have 100s of these in lab
e. EcoR1 and Sma1 are examples of these enzymes
i. If you look at EcoR1 this sequence at the top.
1. Read the top and bottom strand from opposite directions
2. It gives you the same sequence and this is called a Palindrome sequence
f. Restriction enzymes recognize palindromic sequences and it cuts this DNA at this exact position
g. EcoR1 cuts DNA There to give you these two DNA strands
h. Sma1 is another example that cuts this sequence CCCGGG.
i. Difference in these two enzymes is the first give a sticky end or overhang and the bottom gives us a blunt end
j. Restriction enzymes recognized palindromic DNA sequences
k. Some enzymes give rise to sticky ends or overhangs and other enzymes give rise to blunt ends
l. When you cut this DNA with a restriction enzyme you can religate them with a different DNA fragment
m. That is the whole principle of DNA cloning
XLIII. HOW DO WE DO IT? [S43]
a. For example you can take target DNA and again you can go through this but I’m not going to focus on detail
b. You can take target DNA and vectors and digest it with these enzymes and you can ligate or reanneal them
to give rise to a new recombinant molecule and you can form example transform back in bacteria.
XLIV. RECENT ADVANTAGES AND APPLICATIONS [S44]
a. I’m not going to concentrate on that. It’s only for people who are interested.
[End 57:54 mins]