How was the Lac Operon discovered

• Spread a mutagenised bacteria onto petri dish.  Each dot you see is one bacterial colony with millions of siblings. 
• Nutrient medium sontains glucose as the only source of of carbon and Xgal (turns blue if cleaved), but no nducer of the lactose system.  
• Ordinary colonies will be unable to porduce B-galactosidase on medium with no inducere and cleave Xgal
• Only mutants will turn blue

• mutants are isolated
• isolates were mapped with genetic methods
• 3 mutations determined to be I, O, and P
• I and O make B-galactosidase w/o inducer
• All genes in operon (Z,Y,A) whenever I or O was mutated, they went up and down together displaying coordinate control
• P- mutation, all genes were not espressed. 
• Mapping showed that mutations were very close to each other and  just upstream of 3 genes

• Bacteria has an F (fertility) plasmid.  Mobile F plasmid will linearly transfer genes  in fertility plasmid to the bacterial chromosome.  
• F plasmids can be constructed with an different copy of the lac operon to transfer it to another set of genes

• If a locus can function in trans, then it must make a gene product that can diffuse through the cytoplasm and interact with the other genome
• If  the cocus in question functions to control gene expression only in the same genome, then it is operating in cis

• Binds tighter than DNA polymerase
• 2 copies of helix-turn-helix structures on each dimer of the repressor
• Lac I gene binds to 3 sites in operator because nature wants very tight and effeicient binding
• repressor dimer binds with binding sites and effectively puts a loop in the DNA
• Binds with powerful ionic interactions

• well known that lactse operon cannot be extresed in the presence of glucose.  Measure messenger RNA now
• add glucose to mutants, then measure messenger RNA again 
• Dilute glucose and the message will go away

• regulatory mutants were isolated that could not express lac operon in the absence of glucose, even with inducer present
• Found unique mutation in a new control site elsewhere in the chromosome.  First locus encoded CRP and was trans active.  The second (CRP binding site)were close to the operon and were cis mutations
• CRP gene that produces a protein that binds cAMP.  with no glucose, the CRP bound to DNA will stimulate RNA polymerase for lac operon
• With glucose, cAMP levels are low threfore CRP cannot bind to DNA

• Negative control:  a mechanism that prevents transcription via the lac repressor inhibiting transcription
• Positive control: CRP binds cAMP molecular signal then binds to DNA is required for transcription

• Operon region codes for genes araB, araA, and AraD that create 3 enzymes to take arabinose sugar and create energy
• If no arabinose is present, operon is off
• If glucose is added to bacteria with arabinose operon, it turns off
• Upsteam of arabinose operon is araC.  This codes for araC protein.  Protein codes for a repressor with glucose present, but can be activator when arabinose is present

• When araC proten is depleted, the araC gene is transcribed from its own promoter (autoregulatory). 
• When glucose levels are high, AraC binds to both AraO2 and AraI operator sites  This puts a loop in the DNA, repressing araBAD transcription
• When arabinose is present, araC binds with arabinose and changes conformation to become actovator, loop is opened, and araC binds with AraO2 and AraI.  THe operators act in tandem to facilitate transcription of the araBAD gene

• Operon with 5  cotranscribed genes in it
• Each has its on ribosome binding site
• Gene code for enzymes to create tryptophan
• 2 signaling levels that bacteria uses to turn on operon

1. tryptophan repressor that binds to operator. Repressor binds TRP that allows for DNA Binding
2. attenuation

At the front of the transcribed region there is a TrpL or transcript leader region.  I this region are 4 semi-complemetary regions.  1 is partially complementary to 2, which is partially complementary to 3, whic is partially complementary to 4.  Thus, there are 3 possibsle combinations: 1/2, 2/3, and 3/4 to create hairpins.  The creation of 1 hairpin prevents the creation of another.  When Trp levels are high, the ribosome translates sequence 1 and plysically blocks season 2, allowing for the creation of the 3/4 termination hairpin.  When Trp levels are low the ribosome will physically block sequence 1, which allows for the creation of the 2/3 hairpin that blocks the 3/4 termination hairpin

Receptor ligand binds.  Activations happens within the cell which turns on many genes once the protein has been activated

Bioinformatics aligned several bacteria genomes and integenic homology was observed.  This suggested that this DNA may be important.  Analysis suggested that they were flanking promoters and terminators.  There for it is possile that a unique RNA could be made.  RNA was extracted and run on a gel, and probed with  complemetary sequences that determined that the regions were transcribed in-vivo.  Microarray analysis further delineated where the RNA starts and stops

When some bacterial mRNA are analyzed, there ares a long 5' untranslated regions, then the open reading frame region comes.  These folded RNA bind ligand like protein.  They also change shape which  which either regulates the transclation of the mRNA or transcription by attenuation-like mechanisms

The virus integrates due to AT DNA sequences with symetrical arrangements between DNA.  There is alignmet of the DNA, site specific recombination. Genes in both the virus and DNA are responsible for the recombination.  The virus encodes and incesionase and an integrase all the genes to go in and out of integration in this recombinated state.

1. From the promoter left as small transcript is made making the protein N
2. N protein interacts with RNA polymerase with other factors and enables RNA polymerate to bypass rho dependent transcription event so transcription can continue on into downstream genes.  Happen rightward and leftward promoter
3. Create protein Cro, but after any termination, we create critical protein C2
4. For transcription, in late transcription, RNA ploymerase must make antitermination proteins

1. Lysogeny established by expressing integrase, cII (positive transcription factor), and cIII (controls the level of cII) (allows for more CII in healthy cells and represses CII in starvation cells)
2. once that happens the promoter for represser establishement creating a burst of transcription to make lots cI repressor of virus
3. If enough cI is made it will interact with the operator promoter region of the major promoter left and right, shutting them off.
4. If the virus is to remain in the cell, then a perfect amount of cI must be made.  To do this, a special promoter called the promoter for represser maitnance

• Repressor (C1 protein) has the following order of intrinsic affinities for the subsites of O_R:  O_R1>O_R2>O_R3.  Same is most likely true of Operator Left subsites.  At high C1 levels, C1 will bind to all subsites
• Cro competes with prepressor for operator binding sites.  but it does not have an extra domian, so it cannot carry out cooperative binding, lowering its affinity

1. If there is no repressor, we transcribe from promoter R
2. If 1 repressor on O_R1, activity will be turned off
3. If we increase repressor, it iwll occupy O_R2 an interact coorperatively with 2 domiains.  Now the repressor is an activator protein for the promoter for repressor maitnance it turn on.
4. When all 3 sites are occipied by repressor, the promoter for repressor maitnance is turned off

1. expression plasmids were created.  Within a promoter gene was attached to a reporter gene, measure its levels and then you can determine what it is doing.
2. Used the reporter gene LacZ
3. On the same plasmid is cI repressor gene in order to control the levels in the cell system to observe the changes in the reporter genes

• DNA is wrapped around nucleosomes
• Multiple nucleosomes are linked in a beeds on a string formation. 
  
• The beaded nucleosome is condensed into a 30nm fiber. 
  
• The 30nm fiber is looped and condensed. 
  
• There is then high order chromatin folding
  

• more complicated than bacterial cell
• Genes are not organized into a mini genes on 1 transcript
• Genes are combined introns and exons
• can make multiple messages from 1 gene using promoters specific to a particular region
• sequences very tremendously in copy number per genome and can be classified on this basis.  Some encode genes and some do not

• In original cell, there were exons, but bacteria lost them
• They were originally there to promote evolution or to permit evolution under selective pressure
• Exons were shuffled most likely due to unequal recombination events

1. DNA is highly repetitive and is localised around the centromere of the chromosome. 
   
2. It got highly repetitive by uneven recombination events
   

Protein-coding genes may be solitary or belong to a gne family due to uneven recombination

1. they are essential and additional genes are there as a back up to mutation
2. There are genes for infant and adult versions of proteins

1. When there is any damage to the chromosome of the host (ex: UV light)
2. Anytime the overall health of hte host is compormised

1. If DNA is damaged, there is an SOS response.
2.  In bacteria, the protein RecA (recombination protein, and protease) that appears as an SOS response.  
3. RecA cleaves the repressor for lambda which puts it in lytic mode.

1. Transposons (Directly from donor DNA to target DNA)
2. Retrotransposons (proceed through RNA intermediate)

• The sequences all code for transposase that cleaves itself, once aligned, and give a staggared cleave anywhere in the genome
• Once aligned, the 3' of the donor is ligated to the 5' of the target. a 5' to 3 ligation is on the other side.
• DNA polymerase fills in 3' the gaps  
• DNA ligase seals it up  
• many insertion sequences will replicate themselves while they are inserting themselves into a target genome

A insertion sequence that requires an RNA intermediate to inset somewehre else.  2 kinds:

1. viral if there is an extracellular lifestyle (HIV)
2. Non-viral of variable length (LINE, SINE, Alu)

• All retrotransposons have similar structure (suggests ancient)
• Found in all eukaryotic genomes
• inserts at random sites
• can have an extracellular lifestyle

• Ty element used to engineer recombinant Ty in plasmid vectors
• Created a galactose responsive TY and a Galactose responsive ty with an unrelated added intron
• Transform yeat cells gro in galactose and nin galactose containing media.
• In gel responsive Ty, the Ty mRNA synthesis was inc. and the transposition of TY elements inc
• In the gel responsive Ty with an unrelated added intron, Ty mRNAs lack intron and transposed Ty elements lack intron
  

• remain in the genome, but do no have a virus lifestye (no coat proteins, do not infect cells)
• 2 types:
• Long interspersed elements (LINES)
• have protein coding regions with a large promoter
• duplicated via LINE RNA already transcribed into DNA
• Knicks made, priming of reverse transcriptage occurs, then a DNA copy is made, they are aligned, and the insertion is completed using cellular enzymes
• Short interspersed elements (SINES)
• duplication begins with RNA transcription event
• A run of U's and A' form a hairpin loop which can be used as a primer for DNA.
  

• DNA transposons around an exon
• a line insertion even leads to the movement of an exon from one site to another.
  

• Hemophilia
• muscular dystrophy
• apert syndrome
• 20 human diseases attributed to Alu jimping alone

• an alu sequence has jumped into the throbiast growth factor receptor gene.  The insertion has been detected bia PCR of relevent sites in the gene
• Causes gross deformities
• can be passed down if the affected reproduce

• A gene that is there, but does not produce any product

• It is possible that pseudo-gene come abour by the random insertion of cDNA of an authentic mRNA elsewhere int eh genome.  A reverse transcriptase event must have happened to cause this

• In salmonella
• Microbial pathogens can avoid immuno-defenses by using a DNA transposition to change the protein  that the microbe emits.

• The promoter is dead, which prevents it from making the H2 protein and it begins to make H1 protein
• Proter flips in chromosome, and points the wrong way
• hen recombinase causes a complete flip in the promoter, which turns off H2 and turns on H1