CLASS: 11-12 Scribe: Christine Sirna

CLASS: 11-12 Scribe: Christine Sirna

DATE: 10-18-2010 Proof: Louisa Warren

PROFESSOR: Adrie JC Steyn BACTERIAL GENETICS Page 9 of 9

I.  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

II.  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.

III.  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

IV.  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.

V.  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

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?

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

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