Last cellular and molecular genetics exam

Question 1

1. The daf-2 pathway regulates dauer formation and life span in C. elegans. What happens phenotypically and on a molecular level if
   
   1. daf-2 activity is up-regulated (II)
   2. delete daf-2 (II)
   3. daf-18 activity is up-regulated (III)
   4. daf-18 activity is down-regulated (II)
   5. age-1 is deleted (II)
   6. the phosphoinositide-3-phosphate generating kinase is down regulated
   7. the phosphoinositide-3-phosphate generating kinase is upregulated p85/age-1 activity (II)
   8. olfactory and chemosensory neurons are removed, inactivate unc-64 and unc-31 (II)

Answer 1

1. 1. daf-2 activity is up-regulated (II)
      
      • leads to phosphorylation of daf-16 -> stops it from translocating into nucleus -> dauer formation prevented
   2. delete daf-2 (II)
      
      • means that daf-16 will not be phosphorylated and will translocate into nucleus -> increased life span, dauer formation
   3. daf-18 activity is up-regulated (III)
      
      • daf-18 codes for a phosphatase that dephosphorylates PIP3, decreasing PIP3 levels -> leads to daf-16 not being phosphorylated -> daf-16 translocates to nucleus -> increased life span, dauer formation
   4. daf-18 activity is down-regulated (II)
      
      • means that there is more PIP3 -> phosphorylation of daf-16 -> daf-16 not translocated -> no dauer formation
   5. age-1 is deleted (II)
      
      • age-1 will not be able to phosphorylate PIP2 to PIP3 -> no phosphorylation of daf-16 -> increased life span, dauer formation
   6. the phosphoinositide-3-phosphate generating kinase is down regulated
      
      • PIP2 will not be phosphorylated to PIP3 -> no phosphorylation of daf-16 -> increase life span, dauer formation
   7. the phosphoinositide-3-phosphate generating kinase is upregulated p85/age-1 activity (II)
      
      • more PIP3 generated -> phosphorylation of daf-16 -> prevent dauer formation
   8. olfactory and chemosensory neurons are removed, inactivate unc-64 and unc-31 (II)
      
      • insulin not secreted -> daf-2 not activated -> daf-16 not phosphorylated -> increased life span, dauer formation

Question 2

1. Why is telomerase inactive in somatic cells? Which (mammalian) cell types show telomerase activity? (III)

Answer 2

1. Prevents uncontrolled cell proliferation and the development of cancer
2. Germ line cells and stem cells (tumor cells in pathological situations)

Question 3

1. name the five classes of cellular RNA and which ones are processed (II)

Answer 3

1. mRNA
   
   1. capping, splicing, poly(A)
2. tRNA
   
   1. base modifications, nucleolytic processing
3. rRNA
   
   1. base modifications, nucleolytic processing
4. miRNA/siRNA
   
   1. cleavage though Dicer/Drosha and Dicer/R2D2
5. snRNA
   
   1. freed through splicing

Question 4

1. Why are only a few cells malignantly transformed even if p53 fails to induce senescence? What is meant by “first barrier” and “second barrier” against cancer formation? (IIII)

Answer 4

1. First barrier = cellular senescence from normal ways (like p53)
2. Second barrier = genomic instability resulting from too short telomeres that kills cells
   
   1. rapid cell division causing telomere attrition
   2. (is why only a few cells are transformed)

Question 5

1. Name 3 examples of protein(s) (classes) and 3 examples RNAs that need to be transported across the nuclear envelope (II)

Answer 5

1. Proteins
   
   1. In:
      
      1. Transcription factors
      2. DNA/RNA polymerases
      3. Histones
2. RNAs
   
   1. Out:
      
      1. mRNA
      2. tRNA
      3. snRNAs
      4. miRNAs
      5. ribosomal subunits

Question 6

What is a stem cell? What is the difference between pluripotent and totipotent stem cells?

Answer 6

1. A stem cell is a cell with the ability of self-renewal
   
   1. Has the ability to give rise to a cell that is more mature
2. Totipotent stem cells:
   
   1. Also called omnipotent
   2. Can differentiate into embryonic, non embryonic types
   3. Fertilized egg is totipotent, as well as a few divisions from fertilized egg
   4. Can give rise to a viable organism
   5. Can differentiate into all cell types
3. Pluripotent
   
   1. Can differentiate into nearly all cells
   2. All cells derived from any of the 3 germ layers
   3. Can’t make a viable organism

Question 7

1. What is the difference between naturally occurring DNA ends (telomeres) and double strand break generated DNA ends? (II)

Answer 7

1. Telomeres are very rich in positive GT repeats
2. are bound by shelterin complex to form a telosome
3. telomeres are either in t-loop (in vertebrates), or in capped form (in yeast)

Question 8

1. What is a riboswitch, where can it be found and what can be regulated by it? (II)

Answer 8

1. An RNA region in mRNA that changes its secondary structure in a ligand dependent manner
2. Can then regulate the translation of downstream genes
3. Eg. TPP synthesis is regulated by riboswitch in manner dependent on its concentration (negative feedback)

Question 9

1. what process is the tmRNA involved in? describe briefly what other factors are necessary

Answer 9

1. Trans-translation: a ribosome rescue mechanism for non-stop complexes (lack of a stop codon)
   
   1. Ribosome resumes translation using tmRNA as a message
   2. Terminates at a stop codon at the end of the tmRNA reading frame and is recycled
   3. The mRNA and nascent polypeptide chain synthesized/used are targeted for destruction
2. tmRNA (transfer messenger RNA)
   
   1. functions similarly to both mRNAs and tRNAs
   2. Ef-Tu can bind to tRNA like domain
   3. Can tag peptides for degradation
3. Small protein B (SmpB)
   
   1. Associates with tmRNA to stabilize its structure
   2. Enhances affinity for alanyl-tRNA synthetase
   3. Is required for interaction with the non-stop complex genes encoding tmRNA (ssrA and smpB)

Question 10

1. Define the term Hayflick limit. (IIIII)

Answer 10

1. The number of culture doublings in vitro after which cells cease proliferation and are irreversibly growth-arrested, but still alive (senescent)

Question 11

1. during alternative splice what factors lead to the inclusion of exons. Mechanism of proteins and how do they interact with each other/ splicing apparatus

Answer 11

1. splice factors are what regulate alternative splicing
   
   1. positive regulators (often SR proteins) lead to inclusion of exons
      
      1. SR proteins promote binding of U2 and U2AF
   2. negative regulators lead to exclusion (eg. hnRNPA1)
2. for example: sex determination in drosophila
   
   1. different Dsx proteins (produced via alternative splicing) are produced in males and females
   2. 3’ splice site of exon 4 has unusual recognition sequence, leading to it being skipped in males
   3. In females, that express Tra and Tra2, Tra2 binds to 3’ splice site, recruit further splicing factors

SR proteins promote binding of U2 and U2AF

Question 12

1. How do telomeres differ from DNA ends generated by double strand breaks? What is the function of this special structure? (II)

Answer 12

1. Have either a capping model in yeast or a t-loop model in vertebrates
2. Protects the ends of DNA from DNA repair mechanisms that would otherwise mistake it for a DSB (would lead to genomic instability)
3. Also serve as a sort of internal clock for time until cell reaches senescence

Question 13

1. what are 5’ exoribonucleases, 3’ exoribonucleases and endonuclease? Give an example for each. (II)

Answer 13

1. XRN1 = 5’ exoribonuclease – hydrolyze from the 5’ end
2. Exosome complex = 3’ exoribonuclease – hydrolyze from the 3’ end
3. Rnase III, Rnase H = endonuclease – internal cut site

Question 14

1. Give three characteristics of telomere DNA. (II)

Answer 14

1. GT rich
2. 100-1000 bp long
3. 3’ overhang structure

Question 15

1. Many bacteria seem to divide symmetrically. Name three ways to generate asymmetry during division. (II)

Answer 15

1. Starvation
2. Oxidative stress
3. Antibiotics
4. temperature

not 100 % on this answer

Question 16

1. Define the terms „life span“ and „life expectancy“. (II)

Answer 16

1. Life span: the maximum time an individual in a species will live in ideal conditions (genetically determined)
2. Life expectancy: the average time an individual in a species will in given conditions (with real world risks)

Question 17

1. What is the major function of the TOR kinase? How does TOR activity influence lifespan? (II)

Answer 17

1. Major function is in nutrient sensing
2. Is recruited to lysosome when nutrient sensing is active
3. Activation leads to protein translation

Question 18

1. explain the terms exon, intron and splicing (III)

Answer 18

1. Exon: sequence that is present in the mature RNA and encodes the amino acid sequence of a protein
2. Intron: sequence that is not present in the mature RNA/doesn’t encode the amino acids of a protein
3. Splicing: process through which introns are removed from the primary RNA transcript and the exons are spliced together

Question 19

1. Looking for genes involved in aging processes why does it make more sense to analyze mutants with a lengthened lifespan instead of a shortened life span? (II)

Answer 19

1. Phenotypes that lead to a shortened life span can have causes besides aging
2. While phenotypes that lead to a lengthened lifespan need to be involved in aging.

Question 20

1. three main differences between eukaryotic and prokaryotic mRNA

Answer 20

1. Eukaryotic mRNA has 3’ poly(A) tail and 5’ cap
2. Eukaryotic mRNA is spliced
3. Prokaryotic mRNA has Shine-Dalgarno sequence
4. Prokaryotic mRNA has multiple start and stop codons, eukaryotic only has one

Question 21

1. Briefly describe (only keywords!) the three main steps during the process of cytoplasmic RNA localization! At which intracellular location do RNA-binding proteins recognize their target mRNAs? (II)

Answer 21

1. RNA recognition -> RNA binding proteins recognize signal sequence in target mRNAs (usually in the 3’UTR) in the nucleus
2. RNA translocation -> often active transport along motor proteins
3. RNA release and translation

Question 22

1. what is the mechanism of mRNA turnover in eukaryotes? with proteins mediating reaction

Answer 22

1. If mRNA translation is going on, it is protected via circularization, if not it is degraded
2. Regulated via exonucleases such as PAN2/PAN3, and Ccr4/NOT
   
   1. Remove poly(A) tail to allow degradation
3. the cytoplasmic exosome handles it
4. ARE (au rich element) binding proteins in the cytoplasm
5. Ski complex – channels RNA into the exosome
   
   1. Ski2: helicase that makes sure RNA unfolded to go into channel
   2. Ski3/Ski8 – protein interactions

Question 23

1. Eukaryotic initiation factor eIF2 is a prime target for regulation by postranslational modification (in the context of stress response). How is eIF2 modified and what is the consequence? (IIIII)

Answer 23

1. Phosphorylation of eIF2α on ser-51 in response to stress
2. converts eIF2 from a substrate to a competative inhibitor of eIF2B,
3. thus blocking protein synthesis.

Question 24

1. What is the end replication problem and how does telomerase solve it? (IIIIII)

Answer 24

1. with each replication cycle the chromosome ends are shortened due to two factors:
   
   • 3’ overhang on the leading strand is lost (plus regeneration of the 3’ overhang through exonuclease after each replication cycle on the 5’ strand)
   • loss of the regions bound by RNA primers at the 5’ end since they cannot by replaced by the DNA polymerase (needs a 3’ OH to bind to)
2. Telomerase solves this problem by lengthening the 3’ end of telomeric DNA using its own RNA template (5’ end can then be synthesized by the DNA polymerase)

Question 25

1. 3 functions of mRNA localization

Answer 25

1. Localized translation
   
   1. Eg. Translation of β-actin at fibroblast leading edge
2. Asymmetric distribution of proteins to determine cell fate
   
   1. Eg. Distribution of Ash1 in yeast to determine mating type
3. Determination of embryonic axes (asymmetrical distribution of bicoid and oskar mRNA in drosophila)
   
   1. Developmental patterning
4. Avoiding unwanted translation
   
   1. Transport of myelin basic protein mRNA to processes of oligodendrocytes

Question 26

Rapamycin prevents cell proliferation. What is its target. How does it prevent cell proliferation?

Answer 26

1. Rapamycin forms complex with FKBP12
2. This complex binds directly to mTORC1 complex, inhibiting it
3. Inhibition of mTORC1 causes the cell to stop proliferating

Note: Probably a good enough answer, but maybe more needed, don’t know how much detail they want

Question 27

1. What criteria are used to predict microRNA targets? (IIIIII)

Answer 27

1. Complementarity with seed region
2. Conservation of seed region
3. Exclusion of highly structured mRNA regions

Question 28

1. A) What does the abbreviation SIPS mean?
2. b) What is the difference between extrinsic and intrinsic senescence?
3. c) Give four factors that induces for cellular senescence (IIII)

Answer 28

1. SIPS stands for stress induced premature senescence
2. Extrinsic vs. intrinsic
   
   • Intrinsic senescence is due to natural telomere attrition, after telomeres get too short the cell stops dividing
   • Extrinsic is due to extrinsic stress stimuli, such as:
3. 4 factors that induces cellular senescence
   
   • Oncogene activation
   • ROS
   • DNA damage
   • Lack of nutrients/growth factors
   • Improper cell contacts
   • Telomere attrition

Question 29

1. name four sequences functionally important for splicing (III)

Answer 29

1. 5’ splice site (intron begins with GU)
2. Branchsite with conserved adenine
3. Pyrimidine rich region
4. 3’ splice site (intron ends with AG)

Question 30

1. name 3 prokaryotic peptides that induce ribosome stalling

Answer 30

1. Note: ribosome becomes stalled due to absence of a codon in decoding center due to:
   
   1. Stop codon is missing
   2. Stop codon is read through
   3. Stalling on intact mRNA -> cleavage to form non-stop complex
2. oligo-proline
   
   1. is poor substrate as electron donor and acceptor during peptide bond formation
   2. ribosomes become stalled when synthesizing polypeptides containing multiple consecutive proline residues
3. TnaC
   
   1. Blocks binding of Rho by binding to UGA stop codon, inducing translational stalling
   2. Allows transcription of downstream genes TnaA/B
4. SecM
   
   1. Translational elongation stalling to induce translation initiation
   2. Acts as regulator for sec system
   3. If SecA is present, it acts as a force sensor preventing stalling, and preventing transcription of downstream genes (like those of SecA)
   4. If it is not present, then SecM binds and causes stalling, allowing transcription of downstream genes.

Question 31

1. What is the difference between mitotic and post-mitotic cells? What kind of stress are postmitotic cells specifically sensitive to? Why? (III)

Answer 31

1. Post mitotic cells are cells that can no longer undergo mitosis
2. DNA damage, ROS, activation of p53 pathway leading to apoptosis. Since these cells are no longer being replaced, this is not very good.

Question 32

1. what is a MiRtron and how is it processed ? (IIII)

Answer 32

1. miRNA present in the intronic sequence of another gene
2. It is processed through splicing which bypasses drosha cleavage
3. Followed by debranching and cleavage by Dcr-1/Loqs to an miRNA-duplex
4. Cleavage of passenger strand by Ago-2 in the RISC complex gives rise to a mature ss miRNA.
5. short hairpin introns = mirtrons -> function like regular miRNA

Question 33

1. Which nucleolytic processing steps are required to produce an active micro RNA? Please name the enzyme that performs each step (with substrate and product) and the subcellular compartment where it occurs. If parallel pathways exist please indicate all possibilities (III)

Answer 33

1. Cleavage of pri-miRNA by Drosha/Pasha in the nucelus to form pre-miRNA
   
   1. Alternative: splicing of miRNAs containing introns of other genes (mirtrons), cleavage by tRNAseZ of pri-miRNAs attached to tRNA scaffold
2. Cleavage of pre-miRNA by Dicer/Loqs in the cytoplasm to form miRNA duplex
   
   1. Alternative: cleavage of snoRNAs directly by Dicer to form miRNA duplex, cleavage of special miRNAs directly by Ago-2 (miRNA-451)
3. Cleavage of miRNA passenger strand by Ago to form mature RISC complex in the cytoplasm

Question 34

1. Which enzyme creates secondary siRNAs in C. elegans? please indicate wether the primary siRNAs serves as a primer or not and explain one experimental finding that supports your statement. (III)

Answer 34

1. the RNA dependent RNA polymerase generates secondary siRNAs in C. elegans
2. • the primary siRNAs don’t serve as a primer for this reaction, instead the target RNA serves as a template
3. • experimental evidence:
   
   1. introduction of point mutations into a siRNA targeting a specific gene give rise to secondary siRNAs without the point mutation
   2. expression of two distinct trans genes that have complementary UTR regions → introduction of siRNA against one of the trans genes (coding region only) → the expression of the second transgene is also inhibited!

Question 35

1. sometimes miRNAs are generated together with other non-coding RNAs. Please describe one example (not Mirtrons!) and name the factors that mediate the nucleolytic processing step. (II)

Answer 35

1. pre-miRNA hairpin is attached to tRNA scaffold and liberated through tRNAse 2 cleavage
2. alternatively: processing of snoRNAs by dicer to generate functional miRNAs

Question 36

1. 3 pathways of mRNA surveillance in eukaryotes with what mistake in the mRNA induces them
2. How are they fixed (briefly)

Answer 36

1. Non-stop decay
   
   1. Cause: Missing stop codon (or truncated mRNA)
   2. Fix: Dom34/Hbs1/Ski7 -> mediate 3’-5’ mRNA decay
2. No go decay
   
   1. Cause: formation of stable mRNA 2ndary structures that stall ribosome
   2. Fix: Dom34/Hbs1 -> endonucleolytic cleavage of mRNA.
3. Nonsense mediated decay
   
   1. Cause: Premature stop codon
   2. Fix: eRF1/eRF3 (GTPase) -> decapping and deadenylation of mRNA

Question 37

1. Which protein carries the catalytic activity required for target RNA cleavage? Which factor(s) is required to help with the loading of duplex siRNAs (= after the DCR-2/R2D2 complex is bound)? Dies this step require ATP hydrolysis or is loading an exergonic process? (II)

Answer 37

1. Ago2 has catalytic activity needed for target RNA cleavage
2. Hsp70/Hsp90 required to help, requires ATP hydrolysis

Question 38

1. name a bacterial stalling nascent polypeptide and its regulatory function (III)

Answer 38

1. TnaC -> in the presence of tryptophan expression of this peptide prevents translation termination = ribosome stalling -> ribosome stalling prevents rho-binding to the downstream genes (rho would usually inhibit translation) -> tnaA and tnaB genes are translated (production of tryptophanase)
2. Regulatory function: to couple the expression of tryptophan degrading enzymes to the presence of tryptophan.

Question 39

1. what does the term UPR stand for and which factor is regulation on a translational in its context by what mechanism? (III)

Answer 39

1. UPR = unfolded protein response
2. Hac1
   
   1. -> under normal conditions the 3’ and 5’ UTR of Hac1 interact, preventing translation initiation
   2. -> under stress conditions cytoplasmic splicing leads to the activation of hac1 for translation
   3. -> hac1 then functions as a translational initiator for stress response genes.

Question 40

1. explain “primary rRNA transcript” and name the two general processes by which rRNAs are processed (IIIII)

Answer 40

1. primary rRNA transcript is the first transcript formed by the RNA polymerase I while transcribing rRNA genes -> is several rRNAs in one transcript separated by short spacer sequences
   
   1. in eukaryotes: 18s rRNA, 5.8s rRNA, 28s rRNA
   2. in prokaryotes: 16s rRNA, tRNA, 23s rRNA
2. rRNA processing:
   
   1. rRNA precursor assembles with snoRNA enzymes which introduce modifications (such as methylations/pseudo uridylations) that aid in correct folding and assembly of the rRNAs
   2. cleavage of pre-rRNAs facilitated by snoRNAs to liberate individual rRNAs

Question 41

Describe an experiment in detail that demonstrated the limited replicative potential of primary human cells

Answer 41

1. Hayflick experiment
2. Showed the finite lifetime of cultured human cells
3. Showed that normal human fibroblasts will double a finite number of times.
4. Proof for experiment:
5. Mixed equal number of human male fibroblasts with female fibroblasts
6. The male fibroblasts were grown for 40 population doublings prior to mixing
7. Female fibroblasts were grown for 10 population doublings prior to mixing
8. Unmixed cell populations were grown as controls

Question 42

1. Name and explain two reasons why ageing is not a genetically programmed process?

Answer 42

1. Aging could not have evolved as a death mechanism, since would not increase Darwinnian fitness. Because logically, longer lived individuals would create more offspring and thus be selected for.
2. no evolutionary pressure to develop an aging process, since under real life conditions extrinsic mortality before reaching old age much more common than intrinsic mortality and many more individuals die before reaching old age.

Question 43

1. Name two factors that reduce life-expectancy.

Answer 43

1. Important to remember that life expectancy is number of years an individual is expected to live under living conditions, is not the same as life span (expected to live under ideal conditions). Includes real world dangers!
2. Shortened telomeres (GOT lecture 4)
   
   1. Either through mutated telomerase, or haplo-insufficiency, shortened telomeres are a good sign of shortened life expectancy
   2. Can be inherited, even if you don’t inherit the mutated telomerase.
3. Environmental hazards (food availability, diseases, tigers etc.)
4. Genetic diseases, etc. really just anything under real world conditions

Shortened telomere length with successive generations can result in earlier onset and increased severity of a disease – this is known as genetic anticipation

Question 44

1. Do bacteria age? Briefly describe two experiments that support your answer. (IIII)

Answer 44

1. Bacteria can be cultivated indefinitely in vitro
   
   • Have circular genomes, making telomere attrition not a problem
   • Asymmetrical cell division to produce rejuvenated daughter cell
2. Could take one culture, grow it for a certain number of divisions, then put on same medium as another culture that has just started growing and see if either stops dividing first. Hypothetically, they should both grow indefinitely
3. Could also look at “fertility” in older colonies of bacteria vs. younger ones.
   
   • Compare the rate of doubling over time.
   • If there is not aging, rate of doubling should not decrease over time.

Question 45

1. How does an siRNA become incorporated into the RNA-induced silencing complex? Please use Drosophila as an example (it is best understood in this organism) and indicate that determines selection of the preferentially incorporates siRNA strand (the guide strand) vs. the opposite strand (the passenger strand) (II)

Answer 45

1. dsRNA is bound by dicer2 and R2D2, is cleaved into siRNA duplex
2. siRNA duplex is bound by RISC loading complex and passed to ago2 with the assistance of Hsp70/Hsp90 (ATP cleavage required)
3. Passenger strand cleavage by ago-2 induced through C3PO, resulting in mature RISC
4. Selection of guide strand: thermodynamic stability on side with 3’ overhang, 5’ nucleotide, structure

Question 46

1. List the components of the large and the small eukaryotic and prokaryotic ribosomal subunits (III)

Answer 46

1. Prokaryotes (70S)
   
   1. Large ribosomal subunit (50S):
      
      1. 23S rRNA,
      2. 5S rRNA,
      3. 31 proteins
   2. Small ribosomal subunit (30S):
      
      1. 16S rRNA,
      2. 21 proteins
2. Eukaryotes (80S)
   
   1. Large ribosomal subunit (60S)
      
      1. 28S rRNA
      2. 5S rRNA
      3. 5.8S rRNA
      4. 47 proteins
   2. Small ribosome (40S)
      
      1. 18S rRNA
      2. 32 proteins

Question 47

1. explain the nuclear import cycle for proteins (II)

Answer 47

1. Importin (import receptor) binds nuclear localization signals (NLS) in protein
   
   1. Transports them through NPC by interacting with FG repeats of central NPC channel
2. Once in nucleus, it bind Ran-GTp which induces a conformational change and makes importin release its cargo
3. The importin-RNA-GTP complex is imported out of nucleus
4. Once in cytoplasm againm a GAP induces GTP hydrolysis through Ran, inducing conformational change
5. Importin can once again bind NLS

Question 48

1. Name two essential components of the telomerase enzyme. What is the enzymatic activity of a telomerase? (II)

Answer 48

1. The mTERT catalytic domain
2. The RNA template
3. Has reverse transcriptase activity

Question 49

Explain antagonistic pleiotrpy. Give an example

Answer 49

1. Pleitropic alleles (ones that can influence two or more seemingly unrelated phenotypes) with positive effects at an early age could be favored by selection, even if they have negative effects at a later age.
2. This would mean that pleotropic alleles with negative effects (even very minor ones) early on would be selected against, even if they have positive effects later in life.
3. Even small beneficial effects early in life will outweigh a deleterious effect late in life, even if this results in senescence and death.
4. Huntington’s disease is an example of this,
   
   1. Usually onset at around age 30
   2. Dominant allele
   3. No negative selection against this late onset disease

People are likely to have reproduced before even manifests at all

Question 50

1. Name three functions of the nuclear lamina. How are these functions disturbed in HGPS patients and how does this contribute to the premature aging phenotype. (II)

Answer 50

1. Structurally support the nuclear envelope
2. Determines overall shape of nucleus
3. Important for chromatin organization
4. Gene expression
5. In HGPS patients
   
   • LaminA builds up at nuclear membrane
   • Defects in nuclear envelope
   • Incorrect formation of replication forks
   • Incorrect telomere formation
   • Impaired DNA repair by homologous recombination
   • Degradation of nuclear proteins
   • Chronic stress response
   • Leads to loss of tissue and organism homeostasis = aging phenotype

Question 51

1. Name three potential functions for mRNA localization (IIII)

Answer 51

1. developmental patterning
   
   1. through the asymmetrical distribution of cellular proteins
   2. e.g. embryonic axes definition in Drosophila through the asymmetrical distribution of oskar and bicoid
2. cell-fate decisions
   
   1. through the asymmetrical distribution of cellular proteins
   2. e.g. mating type selection in yeast through the asymmetrical distribution of Ash1
3. localized translation
   
   1. e.g. the localized translation of β-actin only at the leading edge of fibroblasts through the active transport using myosin to the end of the actin filaments

Question 52

1. 3 eukaryotic and prokaryotic GTPase initation factors and their functions

Answer 52

1. Prokaryotic
   
   1. IF2:
      
      1. recognizes the fMet-initiator tRNA and complexes it with GTP
      2. stimulates association of 50S ribosomal subunit, leading to the hydrolysis of IF2 bound GTP
      3. this leads to the initiator tRNA being released by IF2 and accomadates into the P-site and initiation complex is formed
2. Eukaryotic
   
   1. eIF2
      
      1. GTPase, escorts Met-tRNA onto 40S subunit
   2. eIF5B
      
      1. GTPase, mediates assembly of 80S from 40S and 60S

Question 53

1. which molecular motors can be used to transport mRNA? please give an example of the name of one motor and its specific cargo mRNA (II)

Answer 53

1. myosin, Dynein
2. eg. Myosin = motor protein, cargo mRNA = actin

Question 54

1. in C. elegans RNAi can be inherited over many generations after initial triggering via double stranded RNA. How is this possible? Name the gene which is normally not involved in RNAi biogenesis but essential for this process (II)

Answer 54

1. generation of secondary siRNAs is done by RNA dependent RNA polymerase using target mRNA as a template
2. RNA dependent RNA-polymerase encoded on the x gene

Question 55

1. Give a basic (minimal) definition of ageing that is applicable to all (ageing organisms. Name three “markers” to describe ageing by this definition. (II)

Answer 55

1. Inevitable time dependent progressive deterioration of physiological integrity with increasing age leading to impaired function and increased vulnerability to death.
2. Classic markers:
   
   • Increased mortality
   • Decline in fecundity
   • Decline in physical performance

Question 56

1. What is “caloric restriction” (CR)? What happens if you apply CR to rodents? Which pathways seem to be involved in CR control? (II)

Answer 56

1. Reducing caloric intake by 30-40% , near starving
2. Mouse life span increases
3. Most likely nutrient sensing and growth factor and insulin receptor pathways

Question 57

1. Localization of mRNAs can be achieved by several mechanisms. Name and describe three of them (II)

Answer 57

1. RNA diffusion and trapping in specific regions
2. Generalized RNA degradation with degradation protection in specific locations
3. Active transport by motor proteins

Question 58

1. please describe the molecular features that define a short interfering RNA (siRNA) (II)

Answer 58

1. dsRNA
2. 3’ 2 nt overhang: recognized by PAZ domain in Dicer
3. 5’ phosphate = licensing for RISC incorporation

Question 59

What is dietary restriction? How does it influence life span in model organisms?

Answer 59

1. Caloric restriction is a situation in which the caloric uptake of an organism is reduced to 30-40% of normal – nearly starving.
   
   1. In mice has been known to extend life span by 40 – 60%
   2. Also show improved lifespan during aging
   3. Disadvantages are:
      
      1. Reduced body weight and muscle
      2. Sensitivity to cold
      3. Increased sensitivity to bacterial infection
      4. Reduced wound healing
      5. Reduced fertility
2. In some model organisms it can increase lifespan by quite a bit
3. Exact way it works not known, but seems to have something to do with insulin/IGF-1 receptor
4. Also potentially TOR kinase, which has an effect on lifespan

Note: Might need to go over this answer again

Question 60

C. elegans go into a stage with extended life span. What is this stage? How is it induced? explain briefly

Answer 60

1. This stage is called the Dauer larval state, in this state, the larva is:
   
   1. Thin
   2. Can move
   3. Do not feed (plugged mouth)
   4. Remain viable for around 3 months
   5. Do not age
   6. Show increased stress resistance
2. Induced by environmental stresses like:
   
   1. Overcrowding
   2. Heat
   3. No food, no water
3. Will stay in this state until environmental conditions change, upon which it wil continue into L4 stage of C. elegans life cycle.

Question 61

How is LaminA processed. How is this different in HGPS patients?

Answer 61

1. In WT cells, LaminA is processed post translationally as follows
   
   1. Farnesylation of the C-terminal CAAX box -> membrane association
   2. The last 3 amino acids are clipped off (Farnesylated C remains)
   3. Carboxyl-methylation of the farnesyl-cysteine by ICNMT
   4. Clipping off of the C-terminal 15 amino acids of the protein
      
      1. Includes the farnesylcysteine methyl ester
      2. Done by ZMPSTE24 protease
      3. Leads to dissociation from the membrane
2. In HGPS patients
   
   1. C => T substitution at codon 608 in LMNA gene
   2. Causes improper splicing, deleting 50 amino acids from LaminA protein
   3. These amino acids contain the protease site for ZMPSTE24
   4. This causes an accumulation of farnesylated and methylated prelaminA at nuclear membrane
   5. Normally only B-type lamins would be there, but now there are A-type lamins there as well.
   6. A similar effect is seen if ZMPSTE24 protease is inactivated
   7. So in summary, it is not the deletion of the amino acids directly that causes incorrect localization, but the permanent farnesylation caused by the deletion of the protease site.

Question 62

What is SIPS? Name two factors that induce it?

Answer 62

1. SIPS is Stress Induced Premature Senescence, or Stress Induced Premature Senescence-like phenotype (but only need the first one)
2. The phenomenon wherein cellular senescence is induced prematurely due to extrinsic stress (such as DNA-damage caused by radiation, or oxidative stress caused by high environmental O2 concentration.)
3. Two good examples would be:
   
   1. DNA damage caused by gamma-rays or UVB
   2. Oxidative stress caused by H2O2 or high environmental O2
   3. Bonus: cell cycle arrest and replication associated DNA damage caused by Hydroxyurea.
4. Some factors that induce it (more than 2, but just choose some to memorize):
   
   1. Oxidative stress
      
      1. High O2
      2. H2O2
      3. Tert-butylhydroperoxide
      4. Homocysteine (I think this is oxidative stress)
   2. Anti-neoplastice Drugs – DNA damage
      
      1. Mitomycin-C
      2. Cis-platin
      3. Bleomycin
   3. Hydroxyurea
      
      1. Cell cycle arrest
      2. Replication associated DNA damage
      3. Stops ribonucleotide reductase from converting NTPs to dNTPs
      4. Inactivates ribonucleotide reductase
   4. Gamma-rays and UVB
      
      1. Radiation induced DNA damage
   5. Too much growth factor
      
      1. Stimulation with cytokines (TGF-B1)
   6. Overexpression of oncogenes
      
      1. Normally stimulate cell cycle, but overexpression has been seen to induce senescence
      2. Raf-1
      3. Ras
      4. E2F1

Question 63

1. Why poses cancer a major challenge to the longevity of organisms with renewable tissues? (II)

Answer 63

1. Need to balance tissue renewing with cancer prevention
2. If have enough tissue renewal to ensure longevity, you enable cancer formation
3. If you suppress cancer formation, you limit tissue renewability and negatively affect organism longevity

Question 64

1. If you generate a daf-2 genetic mosaic will you get a phenotypically mosaic worm or an uniform phenotype? What does the outcome say about the daf-2 function? (II)

Answer 64

1. You get a uniform phenotype
2. Daf-2 works in a non cell autonomous fashion (systemic effect)
3. A few cells that have a daf-2 pathway regulate the phenotype development in the entire organism

Question 65

1. a) Explain the basic concept of the “evolutionary theory of aging”.
2. b) Explain the “antagonistic pleiotropy hypothesis of aging”.
3. c) Give an example for an antagonistic pleiotropic acting gene and explain how its action is antagonistic pleiotropic (III)

Answer 65

1. basic concept: natural selection can only select for mutations that have early effects (fall into the reproductive lifespan), later effects fall into the selection shadow and are no longer selected for → selection pressure decreases with increasing age
2. alleles that have negative later effects are not negatively selected against and they can accumulate in the population, sometimes they are even positively selected for if it is a pleotropic acting allele that has beneficial early effects but detrimental later effects
3. pleiotropic allele: an allele that influences more than one seemingly unrelated phenotypic traits
4. antagonistic pleiotropic acting gene = p53 → early in life for cancer presentation, late in life induction of cellular senescence

Question 66

1. Describe the composition of the bacterial pre-initiation complex. Name an antibiotic that specifically targets the initiation phase of translation and describe its mechanism of action. (II)

Answer 66

1. Pre initiation complex
   
   1. Small ribosomal subunit (30S)
   2. IF1 and IF3
   3. IF2:GTP:fMet-tRNA complex
   4. mRNA
2. Antibiotic: Kasugamycin -> binds to mRNA binding channel and prevents codon anticodon interaction (but don’t need to know this)

Question 67

1. Which class of codons can vary for regulatory purposes with respect to their strength and what position is determining their properties? Name one example of such regulation in E.coli. (III)

Answer 67

1. Stop codons, +4 position is crucial
2. RF-2 gene is regulated via this mechanism:
   
   1. in the presence of RF-2,
      
      1. the weak stop codon is recognized and
      2. a truncated version of RF-2 is translated that is not functional,
   2. but in the absence of RF-2
      
      1. the stop codon isn’t recognized
      2. and only induces a +1 frameshift
      3. resulting in the 2^nd ORF also being translated and a full length functional RF-2 being synthesized.

Question 68

1. what are the final products of hydrolytic and phosphorolytic ribonuclease reactions? (II)

Answer 68

1. Hydrolytic: nucleoside monophosphate
2. Phosphorlytic: nucleoside diphosphate

Question 69

1. Name the decoding factors involved in the termination phase of translation in (i) bacteria and (ii) eukaryotes and (iii) the stop codons that each factor recognizes (II)

Answer 69

1. Prokaryotes:
   
   1. RF1 – UAG
   2. RF2 – UGA, and UAA
2. Eukaryotes
   
   1. All stop codons recognized by eRF1

Question 70

1. what protein factors mediates the repressive action of the mRNA-bound miRNA-Ago complex (exact wording?) what other complexes are recruited and induce degradation of poly-A tail

Answer 70

Note: not sure if this is answer tehy want or not

1. Repressive action of miRNA
   
   1. miRNA-Ago complex bound to a complementary sequence in mRNA
   2. Ago2 can cleave mRNA and it will then be degraded
   3. PIWI family AGO proteins
2. Degradation of poly-A tail
   
   1. PAN2/PAN3 complex
      
      1. Shortening till 100 As are left
   2. CCr4/Not Complex
      
      1. Until 30 As are left
   3. Exosome and Ski complex

Question 71

1. what is the size of the nuclear pore complex (in MDa), of how many proteins does it consist, and what is its symmetry (IIII)

Answer 71

1. size = 125 mDa
2. consists of 30-50 different proteins (also called nucleoporins)
3. 8 – fold symmetry