Genetics Practice Tests

Briefly describe the structure of lac operon and comment on the following diagram including the concepts about repressed transcription, basal and high level of transcription in situations keeping in view consumption of glucose and lactose in a bacterial cell.

• lac operon is a multi-gene sequence controlled by a singular promoter

• codes for enzymes and proteins that aid in brining lactose into the cell (lac y → lac permease) and breaking down lactose (lac z → b galactosidase)

• repressed when there is no lactose to break down

• allolactose is allosteric regulator

• CAP is not bound and the repressor is not bound

  • leads to basal levels of transcription

• occurs when lactose and glucose are both present

  • cell preferentially uses glucose

• CAP promotes the binding of RNA pol. but requires cAMP to bind

• repressor binds to allolactose, blocking its ability to bind to the operator

• CAP is bound and the repressor is not bound

  • leads to high levels of transcription

• occurs when lactose, but not glucose, is present

• cAMP is produced so CAP binds

• allolactose prevents the repressor from binding, allowing RNA pol. to transcribe

• CAP and repressor are bound

  • leads to repressed transcription

• occurs when neither lactose nor glucose are present

• cAMP is produced so CAP binds

• there is no allolactose present so the repressor is free to bind to the operator

  • blocks RNA pol. from transcribing

Describe the purpose and principle of Electrophoretic Mobility Shift Assay?

• studies the interactions between proteins and DNA

• three samples

  • labelled probe (DNA)

  • labelled probe + protein of interest

  • labelled probe + protein + specific antibody

• the samples are run on the gel and if the protein and DNA interact, they weigh more and don’t travel as far through the gel

(A) Dr. Song found a 15-nucleotide sequence located 300 bp upstream of gene X in cardiomyocytes. She suspects the sequence to be an enhancer and did EMSA with known cardiomyocyte specific transcription factors A and B. Observe the following radiographs of EMSA. Was it possible for her to validate the 15 bp sequence as ‘actual’ enhancer (Yes/No)? Comment on the gel images in detail keeping in view sequence-specific binding of specific transcription factors on the putative enhancer.

• no we cannot determine that the 15 nt sequence is an enhancer

• the gel does not show whether the gene has increased expression

• only shows if the sequence interacts with specific protein

• does appear that the sequence is specific to transcription factor A

• top line in lane 3 represents the interaction between the DNA sequence, the sequence-specific protein, and the TFA-specific antibody

  • weighs more than the probe and the protein + probe

(B) Dr. Song found a 15-nucleotide sequence located 300 bp upstream of gene X in cardiomyocytes. She suspects the sequence to be an enhancer and did EMSA with known cardiomyocyte specific transcription factors A and B. Observe the following radiographs of EMSA. Was it possible for her to validate the 15 bp sequence as ‘actual’ enhancer (Yes/No)? Comment on the gel images in detail keeping in view sequence-specific binding of specific transcription factors on the putative enhancer.

• TRB does not bind to the specific sequence

• if it did, there would be a third line in lane 3 where the protein + probe + antibody bound

• the sequence is binding to a protein, but that protein is not TFB as it did not bind to the protein specific antibody

Fibroblast growth factor gene is regulated by GLI3 (a specific transcription factor).

Make a simple drawing of the FGF gene showing the arrangement, from 5ʹ®3ʹ, introns, exons (assume there are three exons), the promoter and enhancers (in all possible positions). On your drawing indicate all the sites where GLI3 can potentially bind, where RNA polymerase II and the general transcription factors would bind and label the start of transcription as +1.

• enhancers can also be found in introns

Carboxy Terminal Domain (CTD) of RNA polymerase II contains seven amino acid long conserved sequence repeated for fifty times. Which of the amino acid should be phosphorylated as a “go” signal for RNA pol II to leave promoter and start synthesizing mRNA.

serine 5

Enlist two main functions of TFIIH

• helicase (unwinds supercoiled DNA)

• CDK-dependent kinase (phosphorylates CTD)

What are the two main components of TFIID?

• TATA binding protein (TBP)

• TATA binding associated factors (TAFs)

Fill the following table using ✓ and x to show presence and absence respectively of the features mentioned.

Observe the following riboswitch. Answer in the boxes provided.

• first gene is ON

• ORF

  • open reading frame

• second gene is OFF

• significance of Us

  • A-U base pairing is unstable, so RNA Pol falls off, terminating transcription

• significance of terminator (hair-pin loop)

  • allows for rho-independent termination because it is an obstacle for RNA polymerase

Fill in the empty boxes with relevant information. Briefly comment on the diagram.

• Boxes

  • DNA damage

  • Inactive

  • selective translation → activates stress response protein synthesis

  • global translation (inhibited)

  • binds to 40S subunit and mRNA

• eIF2 is an initiation factor active when bound to GPT

• when DNA damage occurs, phosphorylation of a-subunit blocks the activity of eIF2B, so it cannot perform the guanosine nucleoside exchange necessary for eIF2 to form the ternary complex

• inactive complex prevents translation initiation of most proteins, but does allow for selective translation of stress-response proteins to repair the damage

Comment on the Gene Targeting Knock-out Models shedding light on neomycin, neor, ganciclovir and tk and fill the empty boxes regarding information about cell survival in the presence/absence of neomycin and ganciclovir

• Boxes

  • killed by neomycin

  • resistant to neomycin and ganciclovir

  • resistant to neomycin, killed by ganciclovir

• Scenario A

  • no recombination of neo r gene occurs

  • mouse is not resistant to neomycin so it dies

• Scenario B

  • homologous recombination occurs

  • target gene replaced by neo r gene giving the mouse resistance of neomycin

  • no tk integrated so the mouse is resistant to ganciclovir

• Scenario C

  • non-homologous recombination occurs

  • mouse gains neo r and tk genes

  • resistant to neomycin but not to ganciclovir

Describe back splicing. What is its advantage? Give three examples

• back splicing occurs when the wrong splice sites are targeted so the wrong introns and exons are removed or not removed

• advantages

  • stability

    • closed loop makes it less prone to degradation

  • translational control with small RNA

    • whilhey are made in small amounts, they can exist in high amounts in cells that need stable RNAs and suffers heavily under RNA degradation that hinders the synthesis of proteins

  • protein diversity

Define alternative splicing? What is its significance? Describe the mechanism by using illustration highlighting Exon Splice-site Silencer (ESS), Exon Splice-site Enhancer (ESE), Intron Splice-site Silencer (ISS), Intron Splice-site Enhancer (ISE), branch point, activator, repressor protein, snRNPs

• one or more gene (pre-mRNA) can be spliced different ways to create multiple mRNA transcripts

• allows for multiple proteins to be synthesized by the same gene

• exon splice-site silencers and exon splice-site silencers repress the silencing at intron and exon splice sites

  • ESS and ISS bind to repressors

• exon and intron splice-site enhancers activate splicing at intron and exon splice sites

  • ESE and ISE bind to activator proteins

• branch point is where the 5’SS end of the intron binds after the first transesterification

  • forms an intron lariat loop which will be degraded after the second transesterification

  • bulged adenosine

• snRNPs are the ribonucleoproteins that identify the splice sites and perform the catalysis of the introns

Descibe two fates of mRNA targeted by miRNA

• translationally repressed

  • synthesizing minimal to no proteins

• degraded

  • nucleotides may be cut which destabilizes the mRNA

Describe biogenesis of miRNA in terms of steps/intermediates in the nucleus and cytosol

• miRNA is endogenous

  • encoded for in the genome and translated into pri-mRNA

• DROSHA cleaves the pri-mRNA to make smaller transcripts called pre-MRNA

• pre-MRNA transported from the nucleus to the cytosol

• in cytosol, it binds to RISC

  • has an argonaut subunit with slicer activity

  • separates the double stranded pre-miRNA into two single-stranded fragments

• one is degraded and the other is mature miRNA and is used as guide RNA

Ribosomes are ribozymes have A, P, and E sites. Which part of eukaryotic ribosomes provide peptidyl transferase activity?

23S rRNA (often near the P site)

Enlist two types of RNA editing mechanisms

• U insertions/deletions

• C → U

• A → I

Enlist two tumor suppressor genes. Describe the mechanism of action highlighting how they affect cell cycles

• pRB

  • represses formation of retinoblastoma tumors

  • controls the cell cycle by requiring a specific transcription factor to activate the expression of S-phase genes

  • controls the transition from G1 to S-phase (replication

  • CDK phosphorylates pRB which causes a conformational change, loosening EF2’s grip on pRB

  • more phosphorylation causes EF2 to fall of entirely

  • EF2 acts as a transcription factor and promotes the expression of s-phase genes

  • allows the cell to move from the G1 to S phase

• p53

  • regulates the cell’s ability to move from the G1 to S phase

  • activated when DNA damage is recognized

  • activated p53 then acts as transcriptional repressor and inhibits the expression of CDF-21

  • stops the cell in the G1 phase

  • the cell must repair the damage to inactivate p53 to allow the transition into the S phase

What are restriction endonucleases? Give any two examples. Illustrate sticky and blunt ends.

• cut at restriction sites to form restriction fragments

• BAMHI

• Sma I