Mark Isalan Flashcards

Question

Outline the key players in DNA mismatch repair in E. Coli

Answer 1

Mismatch repair system in E. Coli is called MutHLS Three enzymes: 1. MutS recognizes bulges 2. MutL is a helicase that walks along DNA (looping it), until it finds a methyl group so it can recognizes parental from daughter, 3. MutH is a endonuclease that cleaves and nicks the new strand.

Answer 2

Dam methylation is essential for mismatched repair in E. Coli --\> conditions for repair shown in table Table highlights how methylation of the strands directs repair What about the last two rows? Used a virus (PhiX174) to probe this system 1. This virus lacks GATC sites so it doesn’t get methylated or cleaved by enzymes in the E. Coli cell 2. But when we introduce GATC sites we achieved directed repair of the new strand during replication

Answer 3

MutHLS Mismatch Repair Mechanism 1. MutS – moves along the DNA strand until it recognizes a mismatch (bulge) 2. Recruitment of MutL and MutH a) MutL (translocase) binds to MutS --\> Translocase with ATPase activity and forms DNA loops, walking along DNA looking for hemi methylated Dam sites close to mismatch to allow for strand discrimination b) MutH (endonuclease) can then bind to MutL which can guide the cleavage on the opposite side of hemi-methylation (allows for discrimination between New and Old) Note - Cut site can be away from the mismatch (12-13 nucleotides) either upstream or downstream --\> doesn’t matter as the entire region is removed (stochastic) --\> the presence of the MutHLS complex indicates the presence of a mismatch

Answer 4

Once the cut has been made exonucleases are used to excise the mismatched region --\> the kind of exonuclease will depend on the nick is made upstream or downstream of the bulge. Either end up with an exonuclease eating away at the DNA.. 1. 5’ to 3’ direction - Nick is made upsteam --\> Exo 7 or Recj 2. 3’ to 5’ - Nick is made downstream --\> Exo 1 The gap that is created filled by DNA Pol III and sealed by ligase. Note - Exonuclease is used as we are chewing away from an end of DNA (nick)

Answer 5

Instead of CH₃ (methylation) groups you are looking for strand breaks The newly replicated strand is distinguished from the parental strand because it contains strand breaks since eukaryotic DNA contains many replicons it will have many strand breaks due to... 1. Leading stand --\> no Okazaki fragments but there are multiple replication initiation points --\> creating nicks that can be recognised at each replicon. 2. Lagging strand --\> presence of Okazaki fragments means that the daughter strand will have many breaks

Answer 6

Eukaryotes --\> MSH complex responsible for mismatch repair MutS MutL Helicase Exonuclease

Answer 7

Mutations in hMsh2 and hMlh1 genes are a cause of inherited non-polyposis colorectal cancer Affects 1:200 - Causes ~15% of UK colorectal cancers.

Answer 8

When double stranded breaks occur we can have one of two things happen: 1. Non-Homologous end joining - NHEJ 2. Homologous Recombination - H.R. Note – Normally Mismatch repair is not used as this happens after replication whereas H.R. happens during.

Answer 9

Double strand breaks can occur due to DNA damage There are many factors that can cause a double stranded break (Physical/Chemical factors) - Frequently occur during **DNA replication.** Note - Apart from Physical/chemical factors we also have nucleases that come in and cut DNA

Answer 10

1. DNA damage leading to a nick in the DNA backbone --\> during DNA replication DNA polymerase action is forced to stop --\> thus creating a gap or dsbreak in the newly synthesized daughter DNA Image context - Remember that there is going to be another replication bubble which will replicate towards that gap 2. DNA damage causes DNA lesions --\> Results in blocking DNA polymerase during synthesis which will stall at the lesion --\> We are left with a single stranded region that is more prone to breaking --\> increasing the likelihood of a full dsDNA break

Answer 11

DNA double stranded break --\> What know? One method of repairing the double stranded break --\> **Non-homologous end joining** (Simplest Outcome) NHEJ - When a series of proteins bind and stabilize the DNA ends and bring them together to allow them to rejoin and re-ligated. More and more evidence is showing that bacteria are able to carry out such processes.

Answer 12

1. DNA double stranded break 2. Ku (Ku70/K80 heterodimer) is a protein that binds to DNA double-strand break ends - high affinity for DNA ends - sequence independent since it binds to the sugar backbone - ring shaped structure that can accomodate DNA - Note that Ku is evolutionarily conserved from bacteria to humans 3. Ku70/K80 heterodimer binding leads to the recruitment of DNA-PK_cs - DNA-dependent protein kinase, catalytic subunit --\> Activation of kinase activity --\> Shown to lead to the formation of a synaptic complex that holds the two ends of the broken DNA molecule together 4. Depending on the nature of the break, different DNA end processing enzymes may be required, including those that resect DNA ends, fill in gaps, remove blocking end groups, and make the ends ligatable. e. g. Artemis, PNKP, APLF, Polymerases μ and λ, etc. 5. The final step in the repair of a DSB is ligation of the broken ends by DNA Ligase IV 6. Dissolution of the NHEJ complex (Anthony J. Davis and David J. Chen, 2013) 5. 4. S

Answer 13

Yes, it is an error prone process Quite often we end up with flaps that associate incorrectly due to microhomologies (as shown) or the ends fold over forming hairpin or even sometimes reverse Hoogsteen base pairing. e.g. GGG/G forming triplex and quadruplex structures --\> source of slippage when DNA polymerases passes - resulting in base deletion (extra flaps are resolved/removed) or addition One would have to predict knockouts by examining on a case by case basis.

Answer 14

Good - or evolution - Increase diversity Bad - Mutations may reduce the fitness of the organism

Answer 15

Immune system - antibody production Some cells exploit this high mutation rate --\> cells involved in antibody production use (Known as VDJ recombination) --\> increase the rate of mutation in antibody genes - creates new mutants - makes us more resilient to invaders Note - Modern genome editing technology (e.g. CRISPR/Cas9) to make targeted mutations and knockouts  NHEJ is used after CRISPR makes a break in the DNA.

Answer 16

Homologous Recombination (HR) --\> refers to the exchange of DNA with homologous DNA - can be used to repair double stranded DNA breaks. Classic way of looking at it is homologous recombination between chromosome in meiosis BUT! It can also take place in the cell between homologous DNA - remember we have two copes! Idea - we are missing a piece of DNA --\> Where can we get the correct DNA? Homologous DNA acts as a template

Answer 17

H.R. essentially lines up homologous DNA (readily found at the replication fork and swaps it between strands - relatively error free. Variations of homologous recombination can occur to maintain the replication fork but two examples are... 1. **Fork regression** --\> lesion blocks polymerase --\> end up getting the single strands coming off and priming on each other --\> creating extra copies of homologous DNA 2. **Strand invasion** --\> Blue/Red strands are complementary to opposite strands so they can act as primers --\> meaning that one strand can rip off and act as a primer for the other strand - So homologous strand invades and acts as a template for missing region

Answer 18

Basically, H.R provides a general mechanism for repair where intramolecular template information has been lost Used in a variety of situations... 1. Double strand breaks --\> most common 2. Lesions bypassed during replication - Repaired 3. In the Long term - source of evolution --\> due to imperfect recombination leading to parts of the genome being duplicated/deleted.

Answer 19

Blue - Parental / Purple - Daughter 1. Chemical lesion in one parental strand --\> results in missing information during replication as lesion is not copied --\> gap has been created which means that informarion has to be retrieved from homologous DNA. 2. Break open the homologous strand 3. Reform phosphodiester bond so that the genetic information is swapped (Homologous recombination) 4. Anys gaps are then filled in with DNA polymerase and Ligase.

Answer 20

H.R. can occur in a variety of ways but involves the same basic processes --\> as long as there is complementarity (5’to 3’ or 3’to 5’) 1. Single crossover --\> large segments swapped 2. Double Crossover --\> everything between the crossover is swapped. 3. Intramolecular (within the same strand) a) Direct repeats - Deleting everything between the two repeats b) Inverted repeats - Invert the red region --\> red region changes orientation relative to surrounding DNA

Answer 21

1. Ds Break 2. Eaten away by exonuclease RecBCD (5’ to 3’) until you reach chi site --\> this leaves 3’ overhang 3. RecA binds and stabilizes 3’ overhang 4. RecA invades homologous DNA in genome --\> strand invasion allows it to base pair with the homologous DNA - displacing the other strand forming a D loop. 5. Once homology has been found - you have to nick and cut homologous strand using enzyme (allow for strand exchange) 6. Fill in and ligate the nicks that have been formed - swapping the strands 7. End up with 2 homologous strand that are paired up with a cross over between them which is known as a holliday junction (unstable structure that can migrate) 8. Holliday junction can migrate (Branch migration driven by molecular motors) - when this happens the DNA is swapped --\> swapping genetic info --\> fundamental mechanism of HR 9. Once missing DNA is exchanged - Holliday junction cut and re-ligated --\> Known as resolution (random process – endonucleases that come in and cut the crossover region)

Answer 22

Homology search and strand invasion - RecA protein structure carriers this out 1. It can bind to single stranded DNA (3’ overhang) 2. Not 100% understood how it mediates strand invasion but we know that the first step is triplet formation. 3. For triplet formation - Homologous DNA needs to be unwound --\> short stretches doesn’t require much energy but longer stretches do require ATP hydrolysis. 4. RecA coats DNA but allows enough space so that the DNA bind to it complementary homologous DNA 5. Once homology is found, homologous DNA is nicked and ligated --\> forming holiday junction --\> allows for branch migration and geentic information exchange Note - Helical nature of RecA filament means that it can form a triplex structure with a homologous DNA duplex

Answer 23

Yes, it can be used for either single or double crossover Note - double crossover is possible but hasn’t been studied as much

Answer 24

Holliday junction During strand invasion, the invaded duplex is nicked which creates 2 x 3’ –OH strand which can then recombine with the opposite 5’ to 3’ homologous strand This junction between strand (crossover) is known as the Holliday junction Junction is a dynamic structure - moves along DNA unwinding in one direction and reforming in the other. In the middle the DNA is not base-paired as they are being exchanged

Answer 25

The Holliday junction stabilized by recombination proteins (RuvA/B) which are also essential to drive branch migration  which promotes exchange of strands.

Answer 26

We know that both RuvA and RuvB stabilize the junction but more specifically... 1. RuvA - flat structure that binds the 4-way Holliday junction --\> A hydrophobic ‘pin’ in the middle helps to separate the strands as it is a lubricating hydrophobic pin in the middle as the DNA backbone is hydrophilic (charge) - Energy dependent process (ATP) 2. The RuvB ‘motors’ bind either side of the RuvA complex and uses ATP to translocate DNA, opens it up and thus allows genetic information to be swapped.

Answer 27

How much migration? --\> random/stochastic process – creates genetic variation

Answer 28

Resolution of the holliday junction refers to the removal of holliday junciton In order to return to original state --\> There are two options.. Cut _Horizontally_ or _Vertically_ --\> leads to two outcomes.. Horizontal cut --\> also known as gene conversion (one gene jumps from one to another) - patches Vertical cut --\> Also known as crossover - information from one chromosome is swapped with the other chromosome – splice Check

Answer 29

RuvC is the endonuclease in Bacteria - other organism have different endonucleases. RuvC is responsible for the cleavage of the holliday junction Position of RuvC binding and cleavage delineates/decides the outcome --\> It is not known how this is coordinated between the patch and splice variations --\> we think it’s a random process but there is a difference between different cell types.

Answer 30

After cuttting the holliday junction we get Heteroduplexes (DNA that has inherited DNA from other DNA regions)

Answer 31

Process is prone to getting bulge/mismatches --\> mismatch repair is used to repair any errors

Answer 32

1. Contributes to much of the variation in offspring --\> meiosis - gene shuffling --\> Scrambles the genes of maternal and paternal chromosomes leading to non-parental combinations 2. Forms physical links between homologous chromosomes to allow chromosome alignment during meiotic prophase 3. Evolution – horizontal gene transfer --\> i.e. bacteria taking in foreign DNA 4. Important in DNA repair --\> Alternative way to bypass lesion 5. Exploited in biotechnology: genome editing with CRISPR/Cas Note - H.R. is conserved in almost every single cell --\> exceptions very minimal organisms.

Answer 33

Yes! Note that this time we get a double stranded break allowing for two overhangs to invade the homologous DNA Forms a Double crossover during Meiosis since there are two branch points Used to create offspring with different DNA combinations

Answer 34

No! All these mechanisms of DNA repair may occur simultaneously However, whether or not they do occur we can end up with different outcomes as shown in the attached image Hypothetical... Following recombination there is a possibility of a mismatch, two situations... 1. Mismatch repair is repaired --\> DNA sequence is restored to parental sequence - Homozygous ‘A’ 2. Not repaired (happens by random) --\> Changes will persist and following recombination will be inherited by one of the daughter progeny --\> Heterozygous

Answer 35

Specific recombination mediated by viral elements - Similar mechanism to normal H.R. --\> uses enzymes called recombinases Recombinases recognize sequence specific targets that are homologous and allows for recombination. There are two main outcomes... 1. Inverted repeat changes orientation 2. Insertion or deletion which either adds/removes DNA. Result of reaction is dependent on the orientation of the repeat sites

Answer 36

1. Site specific Homologous DNA region that is targeted is very short i.e. 10/20 B.P --\> Enzymes only recognize these specific homolgous sequences 2. H.R. targets larger regions --\> Basically you don't need specifc homologous regions --\> Enzymes function more generally

Answer 37

Integrases are an important class of transposition enzymes - Which allow for Site specific recombination

Answer 38

1. Site specific Att sites (homologous regions) POP’ (phage) and BOB’ (Bacteria) line up with Lambda phage integrase Note the region that is Homologous is ‘O’ - present in the middle of the Att sites. 2. Lambda phage integrase allows for the crossover the of DNA 3. Leads to the insertion of the Phage plamsid Apart from the integrase itself - The process also requires some enzymes from the host cell to complete the process

Answer 39

No! No ATP hydrolysis needed as energy is transduced using tyrosine in the enzyme active site --\> traps energy in a high energy phosphate intermediate --\> drives final crossover

Answer 40

Yes! Each recognizing specific homologous sequences Example - Cre recombinase from yeast

Answer 41

Transposable genetic elements --\> Transposons (‘jumping genes’) are mobile genetic elements that move randomly around the genome Sequence homology is not required - Known as ‘selfish DNA’ Different types: 1. Simple transposons contain only the genes required for their transposition (transposases) --\> like mini viral like elements 2. Complex transposons carry other genetic information e.g. antibiotic resistance Note - Transposases similar to recombinases - cuts and swaps DNA around.

Answer 42

1. Bacterial genomes are parsimonious (efficient) 2. Organized into operons 3. No gaps - No introns 4. Regulation/co-regulation - highly organized Why? Explained by increased rate of evolution --\> due to their short life cycle (20 min) - allows for genome to evolve quicker --\> optimizing it

Answer 43

Eukaryptic genome tends to be a lot Bigger than the Bacterial genome Humans - About 3,234 Mb in length E. coli has roughly 3 Mbp (1Mbp – 1 million base pairs) On average - Eukaryotic is 1000x bigger than E.Coli Generally, eukaryotic genome is considered big and ugly  making it much harder to understand

Answer 44

Upon intially sequencing there was thought to be 20,000 protein coding genes But... Nowadays, there is thought to be 18,000 protein coding genes to create human --\> as many genes turn out to be pseudogenes (aren’t expressed and made)

Answer 45

1. Small genes can be hard to locate. 2. Rarely-expressed genes are hard to detect via Expressed Sequence Tags --\> basically we normally examine RNA transcripts to identify genes but it the gene is rarely expressed this makes the gene hard to detect. 3. It turns out that many genes produce functional RNAs instead not proteins 4. Gene density surprisingly low resulting in large number of gaps --\> many gaps have unknown function 5. Alternative splicing gives protein diversity rather than raw open reading frame number (i.e. like in E. Coli) ---\> more difficult to determine the different splice variants

Answer 46

Non-coding repeats make up at least 50% of the human genome

Answer 47

Karyogram shows chromosomes - all 23 pairs (Mother + father) - diploid 1. Ordered in size 2. Shows banding - known as G-banding --\> Tryptic digest then Giemsa stain a) Dark bands correspond with AT rich regions b) Light bands correspond with GC rich regions Banding pattern can be used to... - identify unique patterns of bands on each chromosome, which can help... - identify chromosomes - diagnose chromosomal rearrangements

Answer 48

During prophase... replicated DNA comes together to form chromosome... Which is formed of two sister chromatids held together at a constriction point called the centromere.

Answer 49

No, Not really --\> chromosome number has nothing to do with the complexity of the organism --\> There is no particular reason for 23 Chromosomes, we assume that the numbers are simply evolutionary accidents One pattern - More DNA in cell --\> correlated with bigger cell --\> allows for the production of a more robust/metabolically active cell - Prokaryotic cells normally have a smaller genome --\> E. Coli 3 Mbp

Answer 50

1. B chromosomes are extra (supernumerary) chromosomes to the standard complement that occur in many organisms - not essential for life Originate in a number of ways including derivation from autosomes and sex chromosomes in intra- and interspecies crosses - Example in deer. 2. Holocentric chromosomes – the entire chromosome acts as a centromere. Best known example C. elegans 3. Extrachromosomal DNA  Plasmids  Organelle DNA in mitochondria/chloroplasts  important for function

Answer 51

Yes - Mitochondria & Chloroplast genomes 1. Mitochondrial DNA (all eukaryotes) --\> Covalently closed circular DNA (rarely linear, e.g. Chlamydomonas) In humans: 16,569 bp, only 37 genes with only 13 protein ORFs. 2. Chloroplast genome (oxygenic phototrophs) - Typically 120 – 170 kbp Single closed circular DNA (rare exceptions), typically codes around 100 proteins, mainly to do with maintenance of chloro and photosynthesis. How did they arise? Mitochondria/chloroplasts --\> evolved from parasitic and symbiotic bacteria that invaded eukaryotic cells. Hence, explaining their own genome. Overtime, they became better symbiotes and lost most of their genome. **Note**: Genes that are lost from the organelle genomes either have been completely lost or migrated to host genome.

Answer 52

Generally speaking, the gene distribution is uneven and differ between eukaryotes But! Generally speaking, we observe a lower density noticeable around centromeres (less dense) Terminlogy 1. **Gene-rich regions** – High density 2. **Gene Deserts** – Low density 2. **Multi Gene Families** – Genes that are related occurring in more than one place (10 different places) 3. **Gene Superfamilies** – 100/1000s of copies of a particular gene spread across the chromosome i.e. zinc fingers

Answer 53

One observation - Gene Density is possibly lower in more “complex” eukaryotes Why? This might be due to more complicated eukaryotes with a bigger body plan --\> need more shuffling around so they need more space to do so --\> However, there is no strong statistical data What evidence is there to support this? a) Introns etc. - “Simpler” eukaryotes have fewer introns e.g. yeast. But no eukaryote has NO introns. b) Many more repeat regions in “complex” eukaryotes

Answer 54

Depends on your definition on complexity It is hard to classify organisms as “simpler” vs. more “complex” --\> debated topic as one could argue that yeast is more complex as it has a more organised/highly evolved genome

Answer 55

Major histocompatibility complex – part of the immune system - codes for cell surface receptor. A 700 kb class III region of MHC located on chromosome 6 which contains 60 genes in 700 Kb + 1 pseudogene (missing start codon). This region also has very high GC content (54%) relative to the 41% over the whole genome.

Answer 56

Some parts are Gene-deserts --\> Defined as 1 Mb with no genes Using this definition there are 82 deserts identified (3% of genome, 144 Mb) - Largest is 5.1 Mb.

Answer 57

25% genome (500,000kb) has no genes. 1. Regulatory regions 2. Gene deserts be present for an evolutionary purpose - drive gene exchange via H.R in order to move one gene closer to another 3. Could these gaps contain big genes (very large genes that span over large areas) - Dystrophin (spans 2.3Mb) --\> big genes have slow synthesis - hard to detect

Answer 58

Genes or gene-related sequences --\> Less than half (38%) of the genome has DNA associated with genes Integenic (Between genes - i.e. not genes associated) --\> 62 % of the human genome is made of intergenic region which is classified into two categories: 1. Unique 2. Repeats

Answer 59

1. Unique - microsatellites (still considered a **repeat** - number of repeats differs between people though) as well unique bigger repeats 2. Repeated - found all over --\> include selfish elements - Lines, SINEs, LTR and transposons - selfish elements acting like viral elements. Non-coding repeats making up to 50% of the human genome.

Answer 60

1. Has a different GC content than the rest of the genome (lower) 2. Present in all eukaryotes 3. Normally categorized by length 4. Can take up roughly 10% up to 50% of genome 5. No coding function 6. Can be separated by CsCl centrifugation due to different physical properties 7. Potentual roles - Some may play a structural role: a) Centromere b) Telomere - regulating mortality of cell  In Vertebrates TTAGGG ~ 2500 times in humans

Answer 61

General way classificaton of intergenic repeats... 1. Tandem Back to back next to each other which are normally caused by slippages by DNA poly 2. Interspersed --\> spread around - separated by big distances normally caused by jumping genes. But.... Some researches classify into 5 categories: 1. Transposon derived repeats (45% of genome) - enzyme associated that allow them to propagate. 2) Inactive gene copies (processed pseudogenes) - RNAs are reincorporated - no regulatory sequence. 3) Simple sequence repeats (2 to 5 nucleotides) 4) Segmental Duplications (5% of genome): Inter- & Intrachromosomal - large chromosomal segments 5) Repeated structural sequences (telomeres, centromeres etc)

Answer 62

Example of Repeated Intergenic Regions in Human DNA - No coding function - Often found in the heterochromatin in centromeres (tightly packed, mainly repeats) Examples: a) α-satellite family --\> 171 bp repeat unit (alphoid DNA, mainly in centromeres). b) β-satellite family --\> 68 bp repeat unit interspersed with 3.3 kb repeat (including pseudogenes)

Answer 63

Tandemly repeated DNA --\> Also known ‘Variable Number Tandem Repeats’ which can be classified into two categories: 1. Minisatellite - 10 to 100 bp, Form clusters up to 20 Kb, Associated with structural features: centromeres. 2. Microsatellite - Even smaller - Can be called ‘Simple Tandem Repeats’/’Simple Sequence DNA’, found in Telomeres (5’-TTAGGG-3’), typically \<13 bp in length (cluster short \< 150 bp), Interspersed with non-repetitive DNA. Very common forms are Dinucleotide Repeats - 140,000 versions in the genome (chromosome 12: CACACACACACACACACACACACA)

Answer 64

1. **DNA replication** Both Microsatellites & Minisatellites are unstable - mutations in DNA repair systems can permit expansion which is a common occurrence in cancer cells. Why is it unstable --\> due to the way that DNA is replicated During DNA polymerase action we can get mistakes --\> priming further back add copies whereas priming further forward removes copies 2. DNA recombination Unequal Crossing Over (prophase I of meiosis) --\> homologous DNA incorrect prime on eachother **Take away message** - You get different number of repeats due to processes such H.R. and replication errors.

Answer 65

The number of repeats vary from human-to-human and are useful in DNA-fingerprinting --\> no 2 humans have the same number of VNTR in one region

Answer 66

Genome Wide Repeats - Transposon derived repeats Characteristics: more than 100bp (some \> 1 kb), Moderately repetitive (\< 10⁶per haploid human genome), sometimes occur as: closely spaced clusters or as dispersed single copies, most copies are in intergenic regions whereas some are in introns. Transposon-derived repeats which can be divided into 4 classes: 1. Long Interspersed Nuclear Elements (LINES) - RNA based 2. Short Interspersed Nuclear Elements (SINES) - RNA based 3. Long Terminal Repeat (LTR) Retrotransposons - RNA based 4. DNA transposons - DNA based Really common - 42 to 45% of Human Genome = 3 types of old retrotransposons

Answer 67

First discovered in Maize by Barbara McClintock Different to other genome wide repeats - No RNA intermediate

Answer 68

Yes, DNA transposition is Mutagenic in nature Its mutagenic cause it causes double stranded breaks and creates mutations at insert site - disrupts genes

Answer 69

**1. LTR retrotransposons** - Fossil of Viruses (Retero viruses) No recent evidence of transposition They have viral elements like elements - long terminal repeat Possible that they propagate in the presence of other retroviral infection - take advantage 2. HERV (human endogenous retrovirus) - inactive and more complete retrovirus in the genome Humans have evolved the KRAB domains that are transcription repressors in order to silence these ERV genes

Answer 70

Junk DNA? - Not true it is not Junk! If it were junk then we wouldn’t have it because it simply becomes a metabolic burden --\> rate of propagation counteracts rate of elimination - select for it Maintenance of this DNA suggest value, for example: 1. transposition an engine of evolutionary change? - mutations 2. Discovering roles for non-coding DNA all the time 3. Micro-RNAs/long intervening non-coding RNAs - control on RNA level. 4. ENCODE shows nearly all genome is transcribed - so it makes sense that they have function.

Answer 71

1. Horizontal - genes jumping from other species/organisms (non-offspring) 2. Exon shuffling - different splicing combinations 3. Duplication and divergence - Low chance of gene duplication (1 % chance for 1 gene in 1 million years)

Answer 72

How gene duplication can lead to a new gene... 1. Ancestral gene has promiscuous activity (i.e. main reaction + side reaction which has no biological function) 2. Overtime the side reaction which had no initial function gains a function (bifunctional) 3. We then get gene duplication and divergence - each gene carry out one of the functions from the bifunctional gene --\> Results in increased fitness

Answer 73

1. Recombination - Unequal Crossing Over in meiosis between homologous chromosomes 2. Recombination - Unequal Crossing Over between sister chromatids in mitosis or meiosis 3. DNA Amplification during replication - Type of errors that occurs in replication 4. Replication slippage - Smallest type of error creating microsatellites 5. Retro-transposition - RNA transposon --\> gene gets transcribed, reverse trasncribed into cDNA and inserted back into the genome

Answer 74

Remember there are two types of recombination - Between Homologous chromosomes in meiosis (they don't pair up during mitosis - no formation of bivalent pairs) or. .. - Between sister chromatids in mitosis & meiosis In both cases... Unequal crossing occurs when similar sequences that are not in the same locus as each other on each chromosome undergo recombination. e.g. repeat sequences that don’t align up properly which is then followed by recombination - resulting in duplication and deletion.

Answer 75

Error that occurs in the replication Bubble during DNA replication DNA recombination with the misaligned repeat regions leading to duplication and deletion - daughter cell will inherit 2 copies or no copies

Answer 76

Replication slippage - Smallest type of error creating microsatellites - Random process - replication is a dynamic process/enzymes are not always present at the stand (they detach) 1. Base pairing breaks/melts due to ambient temperature (bound to happen as its in a dynamic equilibrium) 2. DNA loops out but primes/attaches at the wrong base 3. Polymerase reattaches and starts adding bases - one strand will receive a insertion but the other a deletion - evident after another round of replication. Probability of slippage is also dependent on bases present - AT less hydrogen bonds than GC.

Answer 77

Characteristics 1. Size of duplication ranges from 1 to 400 kb 2. Bias towards centromeres 3. As the average human genes are only 25 kb long duplicated regions tend to carry few genes statistically speaking unless it takes place in a gene rich region. 4. Genome duplication may exchange exons  resulting in new gene combinations.

Answer 78

No Obviously not! On the one hand, it might increase fitness by acting as a source of variation - creation of new genes/functions But Recombination can lead to Genetic Disease... Due to the disruption of gene function Example - Charcot-Marie-tooth syndrome - Physical weakness and difficulty walking - Results from CMT1A ectopic (abnormal) recombination between a pair of 24 kb long repeat elements on chromosome 17.

Answer 79

Under the following circumstances... 1. 1 copy at least retains original sequence 2. Gene gets duplicated and doesn't change - increasing Synthesis rate 3. 1 copy remains selectively neutral - It may accumulate mutations (~0.1% per million years) to form gene relics (‘Pseudogenes’) or become non-functional. 4. 1 copy acquires new function or a sub-function ‘Neofunctionalisation’ e.g. new gene undergoes new/different selective pressures resulting in adaptation to provide a fitness advantage.

Answer 80

Example of Neofunctionalization - New gene function: Trypsin vs. Chymotrypsin Trypsin cuts at Arginine and lysine Chymotrypsin cuts at phenylalanine’s, tryptophan’s and tyrosine’s

Answer 81

Pseudogenes - Characteristics: 1. Not rare 2. Copies of functional genes with altered/missing regions - often contain frameshift/nonsense mutations 3. May serve regulatory roles - antisense RNA strands (RNA-RNA control). 4. Increase genome size (cost/benefit)... Normally speaking, evolution selects for certain things as the benefits outweighs the cost, but it is possible that things (e.g. pseudogenes) are just a by-product of evolution. Nevertheless, the fact that many organism have them would argue disagree with that.

Answer 82

Types of Pseudogenes 1. ‘**Non-processed’ pseudogenes -** **genomic region (DNA) is duplicated to form pseudogene** --\> pseudogene is non-functional due to incomplete duplication or inactivating mutations - not selected for on a evolutionary basis. Furthermore, pseudogene is missing regulatory region. 2. **Processed’ pseudogenes - DNA is transcribed to form RNA.** RNA gets reverse transcribed to form cDNA which gets inserted into the same or different chromosome. Evidence: 1. Lack introns/promotors 2. Contain poly(A) tail/flanking repeats Why does this occur? Random stochastic process - we get random priming allowing reverse transcriptase bind form cDNA.

Answer 83

Yes! More pseudogenes in the human genome than actual genes - roughly 20,000 pseudogenes Many pseudogenes in the ribosomal family (2000 copies) - They are processed pseudogenes meaning that they are created via retro-transposition (LINEs) --\> they have a high expression rate. Ribosomal pseudogenes are highly conserved --\> 2/3rd human Ribsomal pseudogenes shared with chimpanzee genome - Implies possible recent origin

Answer 84

Sometimes Pseudogenes can be activated and regain new functions – 2 types: 1. ‘Neofunctionalised’ - Take on new function - diverging from parental genes 2. Subfunctionalised’ - Taking on a subfunction of parent gene Example of pseudogene with Neofunctionalisation - Makarin1-p1 Pseudogene RNA is used to affect expression/regulation of original parent gene --\> when Makorin1-p1 is expressed it is used to stabilize short transcripts of Makorin-1 gene (parent gene) How does it stabilize? Titrates out trans-acting destabilizing factor & prevents degradation (prevents binding) - basically Pseudogene thus supports active parent gene. Note --\> when a pseudogene gets reintegrated it can either pick up a promotor in forward or reverse direction - Make RNA (sense) to produce gene of interest or reverse RNA transcript (antisense) which can serve a regulatory function - alter parent gene splicing or degrade target.

Answer 85

Yes - retrogenes Retrotransposition - This normally happens if a retrogene gets reintegrated by chance near a regulatory region --\> over time the gene tends to evolve and become more and more functional (correct reading frame, etc.) Retrogenes defining feature - missing introns Example: Testis specific pyruvate dehydrogenase **Note** --\> when a pseudogene gets reintegrated it can either pick up a promotor in forward or reverse direction - Make RNA (sense) to produce gene of interest or reverse RNA transcript (antisense) which can serve a regulatory function - alter parent gene splicing or degrade target.

Answer 86

Consequence is that the sequence changes slightly.. Resulting... 1. New Gene function 2. Change in stability of any RNA-RNA interactions - when having a regulatory function

Answer 87

When a gene is copied and maintaining the same function Example: rRNA genes (Mycoplasma genitalium)/KRAB domains in Humans --\> All copies nearly identical

Answer 88

Multigene families - groups of genes from the same organism that encode proteins with similar sequences. **Duplication** allows for creation of multigene family

Answer 89

1. **Tandemly located** (back to back): α-globin genes (chromosome 16) & β-globin (chromosome 11) 2. **Dispersed** - e.g. 3 aldolase genes on different chromosomes.

Answer 90

1. Concerted evolution - evolve in a similar fashion/share homology --\> Known as paralogous - chance that they evolve in different ways is unlikely. 2. Ectopic (non-allelic) Gene conversion - occurs when homologous recombination changes some of the members of a multigene family - may convert all or part of a gene’s sequence - results in evolution that is not independent. 3. Allelic Gene conversion - This is when homologous recombination converts one allele to another.

Answer 91

Globin Cluster structure --\> Globin family shows both Co-location of genes in the clusters as well as dispersal of clusters Similar organization in all vertebrates but lengths vary and different distribution of pseudogenes. Changes between embryonic and adult genes?

Answer 92

YiesshHHH --\> natural selection favors genes to take up specific functions --\> e.g. Different affinities for oxygen allowing 1. Hemoglobin has separate proteins domains for O₂storage and transport (with different binding affinities!) --\> allows for cooperative binding and allosteric regulation --\> low affinity at low [O2] and high affinity at high [O2] --\> this support the metabolic needs of larger animals (hence, allow them to evolve/exist) --\> delivery of oxygen to oxygen deprived tissues 2. Myoglobin simply has a high affinity for oxygen --\>makes sense for muscle tissue as they want to hold on to as much oxygen as possible even when oxygen deprived --\> can pick up oxygen from Hemeglobin at low oxygen conc. 3. Human developmental Pathway: 3 stages: embryonic, foetal and adult --\> during different points in the human developmental pathway different forms of haemoglobin (only possible because of gene duplication) are expressed Why is this important? Slightly different hemoglobin between fetal and maternal blood --\> this allows for optimal oxygen transfer - this has to be controlled as oxygen is needed for survival of the fetus but can be toxic at high conc. (oxidizing agent)

Answer 93

Yes! Larger duplications than genes and segments are possible! --\> have affected genome architecture in some species (including microorganisms and larger plants and animals) But.... This is normally refers to duplicating **more than one** chromosome **not** singular chromosome duplication This is because singular chromosome duplication is associated with a host of diseases/disorders --\> Down syndrome (‘trisomy’ chromosome 21), Edwards syndrome, trisomy 18, Patau syndrom, trisomy 13 - Leads to Gene product imbalance and Reduced life expectancy

Answer 94

Yes it is possible! Genome sequencing etc. suggested major metazoan lineages have undergone WGD --\> Organisms have different capacities to cope with this e.g. flowering plant use this as a major mechanism to acquire new properties + source of speciation.

Answer 95

Polyploidy - Multiple complete sets of chromosomes Example Eukaryotes contain 2 Haploid gene sets (2n) - 'N' number of chromosome + number before = ploidy

Answer 96

It is thought that ≤ 80% of flowering plants species originated via recent polyploidy --\> examples include: Oats, cotton, potatoes, bananas, coffee, sugar cane, peanut, apples Useful as it Generates new properties - Larger genome results in large cell size - not really understood why But... It turns out that polyploidy can also occur in vertebrates --\> More common in invertebrates, fish & amphibians but rare in mammals.

Answer 97

Two main scenarios 1. **‘Autopolyploidy’** (‘Auto’ = same) - Due to mistake (meiosis error) in forming gametes the cells aren’t fully reduced (production of diploid gametes) - upon fertilization --\> result in new ploidy If its viable can lead to speciation - 1-40% frequency of formation. Theoretically, Endoreplication can also be a cause of polyploidy in autopolyploidy - replication of the genome without going through mitosis 2. ‘Allopolyploidy’ (‘Allo’ = other) --\> polyploid offspring is derived from two **distinct parental species**

Answer 98

Example driven by a recessive mutation - results high error rate during pollen formation - element of genetic control Consequence of this mutation? During 1st and 2nd division the spindles ends up being in the same direction instead of being at right angles to each other so they pull the chromosomes in the same direction - leading to 2n gametes. Alternatively... There is also a a recessive mutation that affects ovule formation - eliminate 2nd division in meiosis --\> 2n gametes Takeaway --\> Both cases you end up with 2n offspring - if two 2n gametes come together we produce 4n gametes which are fertile.

Answer 99

We are not sure how the mechanisms are controlled, if they are controlled or if it’s a stochastic process.

Answer 100

May induce disease symptoms - Phenomenon known as ‘Genomic Shock’ Characterized by widespread activation of transposons, gene expression (Num. of transcription factors changes) and recombination However, plant species deal with it quite well --\> Hence, Short term it can be detrimental but long term drives evolution.

Answer 101

No! If due to Autopolyploidy --\> you end up with 2n combining with n creating 3n offspring --\> this offspring can not reproduce with the parents Why? Unequal number of chromosomes during cellular divison

Answer 102

Autopolyploids tend to be more viable that Allopolyploids - This is because the genes are similar, each chromosome will have a homologous pair which can then form a bivalent in meiosis.

Answer 103

They are tolerated in first generation but offspring are infertile Example - 2n + n --\> 3n - mature adult plants When 3n tries to reproduce and produce offspring via meiosis - only 2 chromosomes (out of three) can form bivalents --\> chromosomes don’t come together correctly and we end up with unbalanced gametes.

Answer 104

Example of Alloploidy - Triploid Wheat-Rye hybrid - two species Wheat (triploid) produces a high yield and Rye (diploid) has high disease tolerance - useful to combine to produce Triticale Problem! F1 generation has 21 chromosomes - lead to meiotic problems (improper bivalent formation) - infertile Solution? Add chemical colchicine - cancer drug to F1 generation --\> interferes with chromosomes - leads to doubling of F1 gametes Hence upon fertilization of doubled gametes - 21 (gametes) to 42 --\> 42 is even so all chromosomes can pair up we get allotetraploid

Answer 105

Definitions: Monoploid: basic set of chromosomes multiplied (the number of unique chromosomes in gametes). Haploid: set of chromosomes present in gamete (irrespective of species number)

Answer 106

Chrysanthemum genus (Flower) - it Is believed that the single diploid ancestral species underwent 3 whole genome duplications. Of the Chrysanthemum genus you have: Monoploid 9, Diploid: 18, Tetraploid: 36 and Hexaploid: 54 species --\> all form bivalents during meiosis - produce fertile gametes This indicates that the species is drifting away from ancestor --\> behaving as a new species -

Answer 107

After WGD there are Drastic change in gene expression --\> Hence, the cell attempts to achieve chromosome expression balance - meaning that it restores diploid like behavior. This is achieved in several different ways: 1. Genetic control - transcription factors are regulated via feedbacks loops to ensure correct expression 2. Epigenetic - DNA methylation/chromatin reorganization  changes gene expression accordingly. Consequence of doubling the number of genes --\> allows organism to carry new mutations --\> as you already have the wild type - new functions/ sub-functions

Answer 108

Two outcomes... 1. Crashed system - Gene expression not under control, increased transposon activation, etc. 2. Alternatively --\> if the organism is lucky it may lead to new variability which creates new functional cells.

Answer 109

Genome duplication takes place --\> Over time we lose particular genes copies --\> Another Genome duplication takes place --\> Over time we lose particular gene copies Hence, the number of gene copies is not constant - changes --\> hence, making it hard to identify WGD

Answer 110

Example of WGD --\> Hox gene family - homeobox genes involved in animal development --\> they occur in clusters. Hox genes - DNA binding proteins/code for transcription factors --\> turn genes on/off in order regulate development Hox gene T.Fs utilize the Homeodomain Hox gene family --\> provides a very quick way for evolution to change the body plan of an organism Any mutations in the cluster - results in major changes in the body plan

Answer 111

Genome duplication and sequence convergence (evolution) in genes and regulatory regions has resulted in morphological complexity thus allowing complex body plans to be formed --\> Duplication & manipulation of Hox genes allow for variety the spice of life bbbyyyy Example: Insects have 1 Hox cluster whereas vertebrates have 4 - indicating that WGD has allowed for the increased copies of these clusters to exist and to be fine tuned through evolution.

Answer 112

Barriers normally occur in the short term.... 1. Infertility 2. Genomic Shock

Answer 113

Note - They are NOT the same thing!

Answer 114

Note - Exon duplication and Shuffling are different concepts But... They come hand in hand

Answer 115

Illegitimate non-homologous recombination is one-way exon shuffling takes place --\> only requires a short primer/overlap/homology. Structural domains of different proteins are joined together - due to the low levels of homology for this process can occur quite easily even though we are dealing with different genes. Topoisomerases - nicks DNA and ligates non-homologous region --\> leading to the shuffling of exons

Answer 116

Domain Duplication - Similar to Gene Duplication 1. Unequal crossing over 2. Replication slippage + recombination in the replication bubble

Answer 117

Futhermore... Shuffling promotes self-interaction capacity (very common) - Allows formation of homodimeric proteins --\> These protein domains self-interact These domains tend to be stuck in exon shuffling compatible structures --\> leads to formation of families and superfamilies.

Mark Isalan Flashcards

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