PCB4522 Exam 1 Comprehensive Study Guide Latest 2023, Lecture notes of Nursing

Download PCB4522 Exam 1 Comprehensive Study Guide Latest 2023 and more Lecture notes Nursing in PDF only on Docsity! PCB4522 Exam 1 Comprehensive Study Guide Latest 2023 Lecture 1 • Mitochondria arose due to the Oxygen crisis 2.0 bya • Hadean area was hot, everything was boiling • We don’t have a clean phylogenic origin. Our nucleus seems to be from a certain branch from archeabacter, our cytoplasm is not as related its more eubacterial. Our spindle factors are spirochetes, we are a collection of endosymbionts • The blue-green algae were making oxygen which is deadly poison. Polluting the atmosphere that turned the iron beds red. Thus, mitochondria showed up • Snowball earth (600 mya), the entire earth was an ice sheet the oceans froze over • The burgess shale where you see complex eukaryotes • What is an organism? Elm trees all came from one tree. Organism doesn’t fit every situation • Species is a made up concept. Never a founding member of a species, always a moving dynamic population • Ring species – distribution of a population around a geographical barrier. Smooth gradient around the ring, but at two extreme points they do not mate with each other • Mitochondrial have their own DNA/genomes/ribosomes (they have bacterial ribosomes). Antibiotics can affect your mitochondrial ribosomes but not eukaryotic ribosomes • Archeobacter make cows burp (methane producers) – one clad of archeobacter that looks a little like eukaryotes have TFIIB and Tata Binding Protein (TBP) are some of the central proteins that recognize the start of transcription. The central components of gene expression in us are almost exactly the same as archeobacter • Archeobacter invented histones (don’t have histones 2 and 4) Lecture 2 • Polyteen chromosomes – endoreplication of the genome • Every plant cell has a big cellulose suitcase around it. Much easier to work with mammalian cells. Hela cells seem to be 80% nucleus. Limits what we are able to find out about plant molecular biology • Three genes critical in the transition from archaic primate origins: • 1. SRGAP2 has been duplicated three times o Two out of the four copies of SRGAP2 are no longer active (pseudo genes) o Duplications in the SRGAP2 gene may have played an important role in human evolution o We get it from our primate origins o In Homosapiens it is present in 4 copies o When it was duplicated it was duplicated very poorly, only partially duplicated. Looks almost like a mistake yet it plays an important role in who we are. Something didn’t work right and allows for expanded cognitive capacity o duplications caused: ▪ First duplication: an increase in the length of dendrite stems and an increase in the spines ▪ Third duplication: believed to increase capacity for cognitive brain function ▪ Third duplication: correlates with the beginning of neocortex expansion in Hominin line ▪ Fourth duplication: slowing down of brain development. Neoteny – prolonging of the juvenile stage. We as tailless apes select for Neoteny for a long time ▪ Temperaments, docility, socializations, are associated with smashed in faces. Inadvertently select for shorter and shorter snouts (those genes are physically associated) • 2. FOXP2 is a transcription factor – it controls gene expression o For a long time they thought FOXP2 was the answer for our intelligence o Related to imitation and language somehow. o Archeologists believe that the appearance of art correlates with the development of complex language o FOXP2 phenotype? Difference between us and the bonobo is in language and imitation area of our brain only on ONE side of our brain. What did we sacrifice for language? Ambidextrous • 3. CNTNAP2 could be the longest gene in the human genome 2.3 million nt o Single nucleotide mutations associated with language impairment, autism, dyslexia, schizophrenia, Tourette syndrome and depression. There are some differences between archaic and modern humans o Mostly introns that is cut out and thrown out o Contains a site for the transcription factor FOXP2 to bind right in an intron ▪ i.e. – bacterial chromosome, eukaryotic chromosome (multiple origins), plasmid, mitochondrial genome, phage, or virus • Archaea deviated and have multiple origins of replication. Bacteria have only one • Once you start DNA replication, you are committed to finish all the way to cell division (with only a few exceptions) • Terminator prevents over-replication in a circular chromosome • For our genes, our trains collide and there are no terminators • rRNAs are structural RNAs. Ribosomal cistrons are the most active genes transcribed on this planet. You don’t want to be doing DNA replication in the opposing direction of big ribosomal cistrons, so there is a terminator in front of them. The genes that are expressed most actively are expressed in the same direction that DNA replication occurs • train wrecks with DNA polymerases are OK, but not when a DNA pol runs into an RNA polymerase head on • Archaebacterial could have more than one replicon per cell. A Eubacteria has one replicon per cell, unless it has plasmids or phages. They are independently replicating DNAs so you could thus have multiple replicons within the cells. However if not infected, typically one replicon per eubacterial cell • Plasmid – autonomous circular DNA genome that constitutes a separate replicon o Single copy control – replicates once alongside bacterial chromosome ▪ Bacterial chromosome, some plasmids, eukaryotic chromosomes. Eukaryotic chromosomes have many replicons; control is a problem; all must active once in cell cycle, but no more o Multi copy control – present in more copies than bacterial chromosome ▪ Some plasmids, mitochondrial and chloroplast DNA • General rules for replication: o 1. Initiation of DNA replication commits cells to division o 2. Division must not occur until after replication is completed • Replication is controlled at the stage of initiation (once you start it there’s no stopping it) o Recruiting the polymerase • Replication fork – point at which replication is occurring (growing point) • Replicons can be linear or circular • Origins can be mapped by electron microscopy, autoradiography & gel electrophoresis • Replication may be unidirectional or bidirectional o Unidirectional replication fork is referred to as replication eye o Replication eye forms a theta structure in circular DNA because it appears like the Greek letter theta o Replication fork movement can be detected by autoradiography using radioactive pulses (growth on one side of heavy label or both sides of the heavy label) • Bacterial chromosome is a single replicon (circular) • Bacterial origin supports: o Initiation of replication o Controls frequency o Segregates replicated chromosomes (segregation is usually associated with the origin NOT in eukaryotes) • Most of life doesn’t use the E coli. Method of methylated DNA to control replication o Methylated on the A (deoxy A methylase Dam methylase puts the methyl groups on) o After replication both new DNA strands are hemi methylated. At this point, you can tell which strand made all the mistakes and which is the original template o You can control the timing of replication if you control the time it takes to get the other strand methylated. Holding off methylation allows better repair if there is mistakes o Dam sites for methylation are delayed because they are sequestered away from SeqA (hides the site from the methylase) at the origin it takes about 13 minutes for the Dam sites to be methylated due to sequestering of the sites from SeqA o Dam- strain can never methylate its DNA o It turns out that if neither strand is methylated replication occurs without control. It’s just hemi-methylated states that delay it • SeqA binds to hemi methylated DNA and is required for delaying replication at the origin and transcription at the DnaA promoter • SeqA may interact with DnaA initiator promoter. The DnaA protein binds and melts the OriC. SeqA has no sequence specificity for OriC suggesting interaction with DnaA gives it binding specificity. When DnaA binds it attracts more DnaA. DnaA has an affinity for fully methylated sites as well as for itself (cooperative building up of DnaA protein at the origin) this binding contorts the DNA so that it melts open allowing DnaB (helicase) to clamp on to one of the strands and begin unwinding the DNA • DnaA binds to specific sequences that say “I am the origin of replication.” Embedded in those sites where DnaA binds sometimes you’ll see the Dam methylation site. DnaA has the opposite binding affinity from SeqA. SeqA likes hemi methylated sites, DnaA likes fully methylated sites • SeqA looks for hemi methylated sites anywhere on the genome. However it has a strange affinity for DnaA so if DnaA is bound someplace it prefers to bind where DnaA is (the origin). When SeqA is bound to other places in the genome it has no effect that we know of • SeqA is leaky and eventually the Dam wins and is fully methylated, so SeqA only delays remethylation • If you have hemi methylated sequences in the promoter sequence that makes DnaA transcription, then replication is delayed • Sometimes the DnaA binding sites overlap the methylation sites. These overlap sites cannot be occupied by DnaA when in the hemi methylated state. DnaA can always bind to sites that do not overlap methylation sites, which gets the ball rolling • Hemi methylated origin is bound to SeqA protein, which is bound to the membrane • After the OriC is fully methylated, the origin is transferred to DnaA, which is also bound to the membrane • DnaA burns ATP and is released from the membrane, the origin is melted open (through unique DnaA dependent melting at the promoter) and now because the strands are isolated, the 6 member DnaB helicase will pull drive the replication fork • DNA replication starts Lecture 3 • In the hemi methylated one of the controlling factors called SeqA binds to hemi methylated GATC sites throughout the genome but especially at that origin because it has an affinity for the controlling protein DnaA • DnaA recruits the helicase, and DnaA also has binding sites at the origin • Takes about 13 minutes to fully methylate the origin, while it only takes about 1.5 minutes elsewhere throughout the genome • DNA polymerase moves VERY fast. Once you initiate replication it goes without stopping • ter sites are directional, they have polarity • Bacterial chromosome is a single replicon o 1. Replication normally terminates when replication forks meet o 2. Ter sites serve as backup to prevent replication from going too far (2 clusters of 5 sites) o 3. Transcription of major genes is in the same direction as replication • Replication, termination and gene orientation in bacteria o 1. ter sites are 23 bp and function in only one orientation ▪ 1. 6 proteins 400,000 Dalton (400k Da) ▪ 2. Must bind ATP before it can bind to the ARS core consensus • ATP changes the conformation allowing it to recognize the origin o Origin can function effectively with a functional A domain and any two B elements o ORC binds to A and B1 domains. Events at A and B1 are critical for initiation • At B3 ABF1 is located. ABF1 appears to be a transcription factor that activates transcription and yet it is linked to DNA replication. AFB1 particularly has been exploited for DNA replication in yeast • Licensing Factor Controls Eukaryotic Replication o With each chromosome containing hundreds of origins, how are they regulated so that the entire genome is replicated, but each origin is active only once? • Licensing Factor Controls eukaryotic Rereplication o The properties of licensing factor: o Licensing factor is necessary for initiation of replication at each origin o It is present in the nucleus prior to replication, but is inactivated or destroyed by replication • Experiment showing need to permeable the nucleus to allow second round of DNA replication (protein synthesis inhibitor has been added  membrane not permeabilized only one cycle) • Replication after first cycle needs cytoplasmic factors to resupply through the permeabilized membrane those used up in the first replication. It can’t undergo but one round of replication if you stop protein synthesis, thus we know some sort of protein must be required to generate licensing factors • G1 is where you will expect the preparation and generation of the license in G1 because next to follow is S phase where you will start “ripping up the ticket” (only fires one time at each origin) • Cascade of events responsible for this: o 1. The ORC is a protein complex (ALWAYS bound to the origin thus it cannot meet our definition of a licensing factor) that is associated with yeast origins throughout the cell cycle (ATP-dependent) o 2. Cdc6 and Cdt1 proteins are unstable proteins that are synthesized only in one G1. These proteins are recruited onto the ORC complex o 3. When Cdc6 and Cdt1 bind to the ORC, they recruit MCM proteins to bind (MCM is the eukaryotic helicase  a necklace of 6 proteins that open up and binds to one strand moving with a particular polarity o MCM (Mini Chromosome Maintenance) moves with the opposite polarity of bacteria o Components of the licensing factor: Cdc6, Cdt1, and MCM Lecture 4 • A replicon is all the DNA that is replicated from initiation at a particular origin. So if you have 3 origins most likely you have 3 replicons • Transcription of RNA is a very thoughtful and highly regulated process, while DNA is get through it as fast as you can making the least mistakes • DnaB has 6 subunits (the helicase that is involved in replication) 6 subunits in archaea, 6 subunits in us. Makes a ring like a necklace then opens up and joins around one strand. As it moves it has a directionality, or polarity. It moves 5’  3’ in bacteria, and 3’  5’ in eukaryotes. • Cdc6 and Cdt1 proteins are unstable proteins that is synthesized only in G1 • DnaB  Bacteria. MCM (2-7)  Eukarya. MCM  Archaea • In bacteria DnaB is a helicase and it moves 5’  3’ • In eukaryotes MCM (2-7) is a helicase and it moves opposite to bacteria, 3’  5’ • In eukaryote MCM is not a homoheximer (each subunit has its own gene) • MCM forms a double hexamer in vitro and exhibits more efficient in vitro helicase activity in this conformation as compared to a monomer o The two replication forks are in close proximity and are stationary • Components of licensing factor system o When replication is initiated Cdc6, Cdt1, and extra MCM proteins are displaced ▪ When MCM is recruited, cooperation occurs and you can sometimes get 30- 40 MCMs. You only need one, and only one remains after the extras are displaced ▪ The degredation of Cdc6 and Cet1 during S phase prevents reintiation (ticket used up) o In metazoans, MCM proteins are in the nucleus throughout the cycle ▪ In budding yeast, they enter only after mitosis ▪ The MCM proteins are now considered part of the “Licensing factor” • The yeast ARS o ORC bound to ori through cell cycle; A-B1 protected against DNase I; hypersensitive site in B1 o Cdc6 synthesized in G1; highly unstable; degraded by ubiquitination. Half-life of <5 min (yeast). Similar story for Cdt1. Cdc6 binds before Cdt1 o MCM complex recruited to the ARS by Cdc6. In yeast these proteins enter the nucleus at G1, in animals MCM proteins are always in the nucleus but only bound during G1 o Once recruited MCM any of the other proteins are no longer needed. Cdc6 and Cdt1 at this point become degraded and ORC is no longer needed but ORC stays there always it’s just not being used any longer at this point • The helicase moves in front of the replicase (DNA pol) because it has to open the strand in front of the moving polymerase. It burns an ATP every time it breaks a base pair  very energy dependent reaction • MCM (2-7) is a hexameric complex with helicase activity. It is loaded onto the origin by the ORC plus ATP • MCM (2-7) is phosphorylated at the beginning of the S phase and becomes active, leaving the origin • CDK (cyclin-dependent kinase) comes in and phosphorylates many of the proteins involved which TARGETS them for degredation (down-regulates MCM activity to prevent second initiation at the origin) • MCM (2-7) is a component of licensing factor o The ORC + Cdc6 requires ATP in order to load the MCM complex onto the origin o ORC, Cdc6, Cdt1 and MCM (2-7) can all be downregulated by CDK activity • The process of MCM function o 1. Parting of the histones (ABF1 is a transcription factor involved in replication which recruits chromatin remodelers and opens up the origin so that it is accessible to the ORC) o 2. Allows recruitment of ORC that can’t bind unless activated by ATP o 3. ORC binds to Cdc6 and Cdt1 o 4. MCM (2-7) is recruited continuously o 5. Geminin prevents further recruitment of MCM (2-7). In multicellular eukaryotes geminin blocks further binding • Geminin disrupts Cdt1-MCM (2-7) interaction and blocks further association of MCM2-7 with the ORC • Cdc6 and Cdt1 are degraded as cell enters S phase • Licensing factor consists of MCM proteins o ORC + Cdc6 hydrolyzes ATP and loads Mcm2-7 onto DNA as a 6-protein ring o ORC initially identifies the origin of replication for Cdc6, Cdt1 and MCM o Synthesis of new H strand. Note: origin for H strand is on the L strand o The RNA-DNA strand is unstable and turns over (1400 bases) o 1. RNA primer synthesized by RNA polymerase. H strand displaced o 2. RNA 3’ end cut (processed) by a 3-strand-specific endonuclease (RNAse) at specific sites o 3. 3’OH extended into DNA by DNA polymerase • D-loop of human mitochondrial DNA o RNA portion of the primer (600 bases) DNA portion of the primer (1400 bases) o Depending on where you get the RNA portion of the primer to stop you will either get it to continue or get it to form a stable primer or a stale Displacement loop that won’t get all of the way around unless in high stress conditions o The primer for replication of the H strand is comprised of an RNA-DNA composite molecule (hybrid) o If RNA primer stops at: ▪ 1st position: It gets elongated into a stable primer ▪ 2nd position: It gets elongated into a stable primer of about 1.4kb ▪ 3rd position: there is nothing to prevent replication throughout all of the genome. THIS is what normally happens. o The stable primers are activated under stressful conditions o The origin at position 57 is used for maintenance mtDNA replication. The arrested 7S D- loop primer is used when the mtDNA is severely depleted. Ethidium bromide inhibits mtDNA replication at 50 nM/ml and leads to depletion o Replication of mammalian mitochondrial DNA has separate origins for each strand o Origin for H strand synthesis is on the L strand o Synthesis of new L strand is delayed relative to new H strand • Mitochondrial DNA D-loops o 1. Single D loop (displacement loop) opening of 7S (~1.6 kb) in mammalian mitochondria o 2. Tetrahymena mitochondrial DNA has 6 D loops at a time (many origins), and plant chloroplasts have 2 o 2. The new short strand is unstable, degraded and re-synthesized to keep the loop open o 3. Starts by synthesis of short RNA primer which may be extended by DNA pol (this is normal for all DNA synthesis) o This illustrates the principle that an origin may be used to initiate a single strand only in the case of D loops. Another example is the rolling circle mode of replication o An origin can be a sequence of DNA that initiates DNA synthesis using one strand as a template • There are two types of independently replicating genomes in bacterial cells: o 1. Plasmids – self-replicating circular genomes that maintain a relatively constant copy ▪ Low copy – from 1-10 copies per cell; have a segregation mechanism – ensures that copies are found in each daughter cell ▪ High copy – more than 10 copies, sometimes up to 100 or more. Are distributed between the daughter cells randomly, doesn’t really need segregation mechanism ▪ Episome – plasmid that can integrate into the host chromosome. ▪ Not a lot of difference between a plasmid and a phage (virus). Some plasmids code a toxin that o 2. Phages – self-replicating entities that produce infectious particles. May be circular or linear. These infect other organisms. DNA or RNA ▪ Lysogenic – phages that can integrate into the host genome ▪ Immunity – the ability of a phage or plasmid to exclude infection or reinfection by a similar entity (makes it so that it can only be infected once) • Phages and plasmids sometimes mediate the exchange of host genes o Sometimes when phages and plasmids exit the host genome, host DNA sequences are taken in the process o When this occurs, these sequences can be transferred to the next recipient host genome o Sex pili starts to depolymerize and draws in contact. DNA exchange happens when they come into contact, NOT through the sex pili, they’re just a connection to draw the two together o For both phages and plasmids, the spread to new hosts is a normal part of the life cycle. For phages this transfer occurs through infectious particles. For plasmids, the transfer occurs directly by a process called “conjugation.” o Both phages and plasmids can be thought of as infections o Free plasmids are always cir3cular, but phages may exist as either circular or linear o In chapter 11 (The Replicon) we looked at how the circular host genomes is replicated. Some phages and plasmids use similar mechanisms; however, there are other ways to replicate a circular genome that produce very high numbers of replicated molecules, something that would be beneficial to phages o Phages (and eukaryotic viruses) often have linear genomes which raises a new set of problems for the replication process o Telomeres are the solution to linear DNA replication where the DNA polymerase can’t synthesize the first few nt’s it sits on o The ends of linear DNA are a problem for replication ▪ Special arrangements must be made to replicate the DNA strand with a 5’ end o The problem of linear replications ▪ Since the new strand is always synthesized in the 5’ to 3’ direction, it is easy to finish the new strand by running off the end of the template, but how does the polymerase initiate at the 3’ end of the template strand/ (How does it get to the 1st base? ▪ DNA pol usually binds region that surrounds origin. It is difficult for the polymerase to start at the end o Strand displacement ▪ Replicate each strand separately ▪ Linear template: Adenovirus • 1. Bottom strand is used as template and the top strand is displaced • 2. Top strand forms terminal duplex before initiating DNA synthesis (inverted repeats base pair to each other and form a loop) • 3. Protein intervenes – often used by viral nucleic acids that have proteins that are covalently linked to the 5’ terminal base. Has deoxy C covalently linked to the protein. Protein binds to the end and the C becomes the first base of the new strand. The protein is forever stuck to that strand. Adenovirus always has a terminal protein bound at the end o Terminal protein requires help from a second protein • Adenovirus starts out as linear DNA. One strand is replicated and the other turns into a free single strand, but DNA pol only recognize double stranded DNA. The inverted repeats on the 5’ and 3’ end of the free single strand fold on each other and form a duplex origin by base pairing to each other. This forms sort of like a loop structure • Strand displacement for linear DNA replication: o 1. The first synthesis uses a normal double-stranded linear template o 2. The displaced strand mimics a linear double strand by forming a hairpin structure ribosome binding site or shine dal garno. If you put a bacteria message into a eukaryote you better remove all the ATGs because the eukaryote will start translation at the first ATG site it finds. • finP does not encoded a protein it encodes an antisense regulatory RNA. finP goes in the opposite direction of TraJ, finP is the negative regulatory of TraJ • TraJ is required it’s made all the time and its levels are regulated by finP • TraY is found at the beginning and TraI is at the end • The Y protein forms a complex with the I protein to form YI • YI is an endonuclease which recognizes the origin and cuts. (analogous to A protein) One subunit recognizes the origin (Y) the other subunit (I) cuts • Y protein goes to find the origin, I protein then goes and cuts the origin. The LAST thing to happen is I protein is made • TraM recognizes when there’s a stable complex formed. Checks off that everything is paired up well. It monitors the pairing to see whether things are locked in place appropriately • TraJ a positive transcription factor • TraY recognizes the origin TraI cuts the origin (work together as a complex) • TraA makes pilin. Pilin is a major protein of the sex pilus. It’s made of little pilin subunits. • The F plasmid is transferred by conjugation between bacteria • A free F factor is a replicon that is maintained at the level of oneIf you put a bacterial message in a eukaryote you better remove all the ATGs • Bacteria look for ribosome binding site, a shine dal garno • FinP encodes an antisense RNA (negative regulator of TraJ) • TraJ is a positive regulator, it is made all the time. It’s levels are regulated by an antisense, FinP • Y protein forms a complex with I protein to form YI • YI is an endonuclease that recognizes the origin and cuts • TraA makes pilin. Pilin is a major protein of the sex pilus. It’s made of little pilin subunits • TraM checks that everything is paired properly • TraJ positive transcription factor • The F plasmid is transferred by conjugation between bacteria. F plasmid  fertility plasmid • A free F factor is a replicon that is maintained at the level of one plasmid per bacterial chromosome • An F factor can integrate into the bacterial chromosome o Its own replication system is suppressed • In addition to be able to undergo bacterial conjugation and transfer itself from cell to cell, when it integrates into the host chromosome, it thinks its transferring itself but since its now a part of the large bacterial chromosome, instead of just transferring itself it has to transfer the whole E coli chromosome before it can get around to where it is located. This process is how you transfer genes from one bacteria to another • E coli actually has restriction enzymes that prevents foreign DNA from coming in. Without this conjugation system E coli would not exchange DNA. It’s always the phage/plasmids idea to party • The F plasmid is transferred by conjugation between bacteria o 1. Bacterial Conjugation: a plasmid genome or host chromosome is transferred from one bacterium to another in a mating process mediated by F plasmid o 2. F-plasmid: an example of an episome in E. coli o 3. Episome: an element that may exist as a free circular plasmid; however, it may become integrated into the bacterial chromosome as a linear sequence also • F-plasmid o 1. Large circular plasmid (100kb) o 2. Only 60% (ca. 60 genes) has been mapped o 3. 32 kb is organized as a unit to transfer its genome to another bacteria (transfer region or tra genes) o 4. Three methods of replication: ▪ a. oriV as free plasmid (one copy/bacterial chromosome) ▪ b. uses E. coli chromosomal origin when integrated (oriC); oriV is suppressed ▪ c. oriT during conjugation • oriV is the normal origin that the plasmid uses to replicate itself • When F-plasmid is conjugated into a different organism it doesn’t use any of its regular origins any more. It’s now a part of the bacterial chromosome, it just uses oriC. OriV us suppressed when integrated, it just used the bacterial OriC • Free F-plasmid o Tra genes – 32 kb ▪ OriT at the start of tra genes o Total  100kb o Tra: Discrete region that has transfer genes o OriV – used to initiate plasmid replication (vegetative – independent replication) o OriT – used to initiate replication for transfer (in conjugation- rolling circle model) • IS elements (Insert sequence) – (simple transposons- encode transposase) o Used to insert into host chromosome – integration o The simplest form of a jumping gene. It’s a mobile DNA element. It’s only interested in its own replication. Most of our genome is composed of these elements which are ineffective and repressed. Most of our genome is comprised of this baggage, 90% o Codes for one enzyme. The enzyme that cuts themselves out that they can jump. Sometimes they’ve been so mutated they’re not functional anymore but they’re still there as baggage o These IS elements have sequences similar to that of the host cell (E coli). The endogenous recombination mechanism of E coli sees that and recombines. And when it recombines, that’s how the F factor gets into the host chromosome. Just by accident o The host didn’t realize it but it just saw two repeated DNA sequences, recombined them, opened up the circle and now the F plasmid is integrated. The plasmid didn’t have to do anything. The F plasmid is only able to integrate because it was itself infected by these IS elements o It became infected by a particular group of IS elements. If the host has any one of these then that’s a substrate for homologous recombination. These IS elements don’t even have to be functional, they just serve as a substrate for a very vital function in bacteria and all organisms: recombination • An F-pilus enables an F-positive bacterium to: o 1. Establish contact by an F-negative bacterium ▪ If a bacterium is F-positive, it will develop immunity to other F-positive bacteria. Signaling that the cell has already been infected (gets hands slapped) o 2. Initiate conjugation by pulling the cells together • Tra region of the F plasmid o Regulation: TraM, TraJ, FinP o TraJ – activates two promoters ▪ TraJ activates promoter of TraM and TraY o TraM – senses that mating pair formed o FinP – negative regulator transcript o TraD (TraN and TraG may also participate in this process) – coupling protein. Makes a connection between 5’ end that’s being pulled off in rolling circle, directing it to the channel so that it can be passed to the next cell. D actually mimics the structure of a natural protein in that channel complex • Ti Plasmid (Tumor inducing) • Agrobacterium/plant interactions o Acetosyringone is produced by wounded plant cells (phenolic compound) – notifies that something is happening with the plant, a caterpillar eating it or something o It turns out that agrobacterium has picked up on this and uses this (Acetosyringone) to induce the transfer of a portion of its Ti plasmid to the plant genome. It genetically engineers a eukaryote to make its favorite food: opines o Agrobacterium in tumor at wound site transfers T-DNA to plant cell o The bacteria genetically modify the plant by inserting 23 kb of genes into the plant nucleus and its integrated and stably inserted causing the plant to synthesize opines, conjugates of basic amino acids, and they can’t be broken down by the plant (constitute up to 7% of the mass of the plant) and this mass of the plant is just pumping out opines where the agrobacterium has a big banquet • Most soil bacteria cannot metabolize opines. The agrobacterium genetically engineers to produce a food source only for itself and not for its competitors • Components of the Ti Plasmid (~200 kb in length) o 1. T-DNA (transferred-DNA): 23 kb region that is transferred to the plant (only the T- strand actually transferred). Ti plasmid contains an oriV, it’s a normal plasmid. There are two main categories of the genes that are transferred to the plant: ▪ a. hormone genes – induce tumors in the plant ▪ b. opine synthesis – plant is engineered grow in an undifferentiated mass and to pump out agrobacterium’s favorite food o 2. Virulence region (Vir): encodes all of the functions required for the transfer of the T- strand to the plant ▪ Took tra, elaborated the genes that were there to function to pass a piece of the plasmid to a plant, to a eukaryotic cell. That means that piece of DNA could act in two ways: disrupt and cause a tumor, or produce opines ▪ Suggested evidence that it actually encoded for genes that made proteins, which means that agrobacterium with no brains/neural network had figured out what a plant/eukaryotic promoter sequence looked like. Sequence the genes, look at the promoter, and find out how these agrobacterium did it ▪ Vir genes, a copy of Tra genes, and now they are responsible for virulence o 3. Opine catabolism: allows the bacterium to use octopine and nopaline nutrient (most other soil bacteria lack these genes). Genes requires to break down opines. o 4. Bacterial conjugation. The Tra region is similar to Tra in other bacteria. This is for transfer of the entire pTi between different agrobacterium • Ti plasmid also carries genes for synthesizing and metabolizing opines (arginine derivatives) o They are used by the tumor cell • T-DNA carries genes required for infection o Part of the DNA of the Ti plasmid is transferred to the plant cell nucleus • The Vir genes of the Ti plasmid are: o Located outside the transferred region (tra) o Required for the transfer process o The Vir genes are induced by phenolic compounds released by plants in response to wounding • The Vir region is responsible for the transfer of T-DNA to the wounded plant cell o VirA (the sensor) and VirG are a part of a two component system. You have a receptor that is usually embedded in the membrane that is looking for a signal. Once that signal is received, it is usually a chemical compound, it causes a modification in the receptor such that it phosphorylates itself. That phosphorylation event triggers a cascade of events which ends up in gene activation. It alter phosphorylates the receptor and then that phosphate is passed on to a transcription factor that now is activated to seek its promoter, in this case, the effector (receptor embedded in the membrane) is VirA and the transcription factor is VirG. VirG becomes the analog to TraJ, which now activates the rest of the regulon o VirB (membrane protein; ATP-binding) – seems to be connected with the channel. Analogous to TraD, leads the tip of the replicated strand to the channel o VirG (the effector) – the transcription factor analogous to TraJ. It is activated to bind to these promoters by interactions with the receptor VirA embedded in the membrane. VirA is activated by the presence of the wound signal, Acetosyringone o VirC (binds overdrive DNA) – modification of conjugation o VirD (1-2) (endonuclease nicks T-DNA D1 & D2) – VirD1 is like TraY, VirD2 does the nicking like TraI. o VirE (2) (ssDNA binding protein. Binds T-strand) – unique because it is a single strand binding protein. In conjugation only a single strand is transferred to the recipient, the same is so with the agrobacterium T-DNA system, and that strand has to be coated with a protein to prevent degradation by host endonucleases • The Vir region is activated by Acetosyringone using a 2-component system o VirA is the sensor. VirG (positive regulator) is the effector, activated by a cascade of events set off by the phosphorylation of the VirA sensor in response to Acetosyringone. ▪ Note: activated VirG causes its own promoter to have a new start point with increased activity o Which genes are constitutively expressed? Those genes that are waiting to receive the signal, such as VirA and VirG o The membrane protein VirA is auto phosphorylated on histidine when it binds an inducer • VirA activates VirG by transferring the phosphate group to it • The VirA-VirG is one of several bacterial two component systems that use a phosphohistidine • Transfer of T-DNA resembles bacterial conjugation o T-DNA is generated when a nick at the right boundary creates a primer for synthesis of a new DNA strand o Only one strand (like bacterial conjugation) is transferred o It has a nick site on the right and on the left portion of DNA to be transferred, agrobacterium have put in two oriT’s. Why? Allows it to cut again making the VirD1- 2 think it’s gone “all the way around” where really it just cut a piece of the DNA o As the rest of the strand is replicated and replaced by ‘rolling-circle replication’ (It’s not really rolling circle because there are two oriT’s so it only cuts off a piece of linear DNA) VirE2 is coating the strand in proteins to protect from endonucleases o The pre-existing single strand that is displaced by the new synthesis is transferred to the plant cell nucleus. Transfer is terminated when DNA synthesis reaches a nick at the left boundary. The single strand is then made double stranded (“by some mysterious process, notice how it looks similar to bacterial conjugation”) o Why do agrobacterium only like wounded cells? Because plants are surrounded by thick cellulose cell walls, very difficult to penetrate. The only way in is to catch it when it’s been wounded and the newly dividing cells have very thin cell walls • Generation of the T-strand o Cut MUST be made on the right border, NOT the left border (the two OriT’s) because if the cut is made on the left border the entire genome will be unwound EXCEPT for what you want to be unwound. Therefore there must be a mechanism to specify cutting at o The single stranded T-DNA is: ▪ Converted into dsDNA ▪ Integrated into the plant genome o The mechanism of integration is not known ▪ T-DNA can be used to transfer genes into a plant nucleus • The T-DNA is imported into the nucleus of the plant cell o The Vir proteins that are transported to the nucleus include: ▪ VirD2 (contains 2 NLS sequences, nicks at border sequences) ▪ VirD1 (as a complex with VirD2) ▪ VirE2 (contains an NLS) • Acetylsyringone is phenolic (hydrophobic) it is permeable to membranes, so it bypasses the periplasmic domain and interact underneath the membrane. Once Acetylsyringone goes in and binds to the part of VirA that’s inside it phosphorylates itself at a histidine. ChvE is another receptor but it’s not embedded in the membrane. There are two conditions that must be met before you get the transfer of T-DNA o An enhancer function that facilitates, the role of ChvE. It binds simple sugars like arabinose, these are the building blocks of cellulose. The cell is wounded, what else do you need to know? That’s it’s going to rise again to form a cell. o 1. Acetylsyringone says I’m wounded o 2. Arabinose says yea but I’m coming back and making a cell wall o With these two conditions met, it’s ideal time for agrobacterium to send bacteria in. ChvE is not absolutely essential but it increases the sensitivity of VirA for very small concentrations of the wounding signal • The phosphate is then transferred from to the effector (VirG) VirG unmasks its DNA binding domain and now the DNA binding domain can go and stimulate the Vir promoters that make up all the Vir regulon • The Vir region is responsible for the transfer of T-DNA to the wounded plant cell o 1. Acetosyringone is produced by wounded plant cells (phenolic compound that is permeable to membranes) o 2. This triggers autophosphorylation of VirA o 3. VirA phosphorylates VirG which causes VirG to become activated o 4. VirG activates transcription from other Vir promoters • The pTi of Agrobacterium has been modified to serve as a vector for modifying the genomes of plants • MiniTi T-DNA based vector for plants o Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene o 1. Binary vector: the Vir genes required for mobilization and transfer to the plant reside on a modified pTi. Do heavy cloning/construction in E coli which can be easily manipulated and grown in lab. Once you get your vector you try to mobilize it from E coli into agrobacterium. Agrobacterium is slow growing and not easy manipulate, so you do your fancy stuff in E coli. Now that you have a cloning vector/vehicle/mobilization plasmid to put into agrobacterium, so you let bacterial conjugation to work for you and to do that o MiniTi – this is what’s used and still used today. Has an oriV so it can replicate, and it can replicate both in E coli, agrobacterium, a variety of bacteria. The it has oriT1 and oriT2 along with overdrive to specify starting at the right flag. It has two other features, one is: ▪ 1. kanamycin resistance (some sort of selective marker). These antibiotics kill the chloroplast and you select for the ones that are still green so you can find the ones that are transformed and those that are not o Contains an insertion site (multiple cloning site) where you can open it up and insert the genes of your source. Right now the technology is limited to one to two genes at a time, but likely we can go much bigger. MiniTi is disarmed such that it has been stripped down and no longer causes a tumor. Taken out everything except for the right and left border and put everything we want in the middle. o 2. Consists of left and right border sequences, a selectable marker (kanamycin resistance) and a polylinker for insertion of a foreign gene