Final Exam Study Questions

Question 1

Describe key features distinguishing group 1 and group 2 intron splicing

Answer 1

group 1 requires an external guanosine cofactor
group 2 requires an internal bulged A for splicing and lariat intron

Question 2

Describe key features distinguishing archaeal and nuclear tRNA splicing

Answer 2

archaea introns are spliced by endonuclease protein (not self splicing like group 1 and 2)
nuclear tRNA intron splicing can occur in the presence or absence of an intron in tRNA genes which defines 2 classes (those that require splicing and those that don’t)

Question 3

Describe key features distinguishing nuclear pre-mRNA intron splicing

Answer 3

spliceosome (large complex of snRNPs and auxiliary proteins) which removes introns and joins exons

Question 4

Describe key features of the composition and function of the spliceosome

Answer 4

dynamic RNA-protein complex which removes introns from pre-mRNA
composition: its 5 U-rich snRNPs and other proteins function: two sequential transesterification reactions and the use of ATP hydrolysis for energy

Question 5

Does mRNA polyadenylation occur before or after termination of transcription by RNA polymerase 2?

Answer 5

occurs after cleavage and release of the mRNA
after the transcription terminates (after RNAP2 transcribes the pre-mRNA)

Question 6

Why is it said that alternative splicing is a mechanism for generating protein diversity from a small set of genes?

Answer 6

because depending on which gene is getting transcribed, the mRNA are different and are including different exons so it’s a way that the same gene can give rise to different proteins in different tissues
allows different combinations of exons to be included in the final mRNA

Question 7

Compare key features of RNA editing in trypanosomes and apolipoprotein B RNA editing in humans

Answer 7

RNA editing in trypanosomes: involves extensive U insertion and deletion in mt mRNA directed by gDNAs to create functional proteins
apoB editing in humans: single, site specific C to U deamination (removal of amino group) in nuclear mRNA so a different protein forms

Question 8

Compare key features of small interfering RNAs (siRNAs) and microRNAs (miRNAs)

Answer 8

siRNA: autosilencing (silencing of the same genetic locus or a similar locus from which they originate), initiate RNA interference
miRNA: heterosilencing (derived from unique genes that specify the silencing of very different genes), target mRNA for degradation or translational inhibition

Question 9

What are “Dicer” and “Slicer”? Which one is a component of RISC?

Answer 9

Dicer: cleaves the hairpin loop structure and ends up a miRNA ds douplex… enzyme that prepares small RNA molecules for gene silencing
Slicer: enzymatic activity of a protein that cleaves target mRNA… the core component of RISC

Question 10

Briefly describe a specific example of a base-pairing interaction between two different RNAs that is critical for translation (i.e., name the two RNA molecules that base pair, and briefly describe their function in the cellular process)

Answer 10

tRNA and mRNA at the ribosome
tRNA anticodon base pairs with the mRNA codon which ensures the correct amino acid is added to the growing polypeptide chain

Question 11

Briefly describe a specific example of a base-pairing interaction between two different RNAs that is critical for RNA interference (i.e., name the two RNA molecules that base pair, and briefly describe their function in the cellular process)

Answer 11

base pairing between siRNA and complementary mRNA which leads to the degradation of mRNA and silencing of the gene
siRNA acts as a guide within the RISC complex which identifies the target mRNA through its sequence, and once the siRNA and mRNA are base paired, the RISC complex cleaves the mRNA to prevent it from being translated to a protein

Question 12

Briefly describe a specific example of a base-pairing interaction between two different RNAs that is critical for RNA editing in trypanosomes (i.e., name the two RNA molecules that base pair, and briefly describe their function in the cellular process)

Answer 12

base pairing between mitochondrial pre-mRNA and a gRNA during U-insertion/deletion editing in trypanosomes
the gRNA provides the editing instructions by base pairing with the pre-mRNA and directing it where to cleave and add or remove U through a series of enzymatic steps

Question 13

Briefly describe the roles of rRNA, tRNA, and mRNA in protein synthesis.

Answer 13

rRNA: structural component of ribosomes, helps to properly position the mRNA and tRNA and catalyzes the formation of peptide bonds between amino acids
tRNA: acts as an adapter molecule by carrying a specific amino acid and matching it to the corresponding codon on the mRNA with its anticodon
mRNA: acts as a blueprint by carrying the genetic code copied from DNA to the ribosome where it is read in three-base sequences called codons, which specify a particular amino acid

Question 14

Provide a specific example of an RNA/RNP for how RNA-protein interactions can influence the catalytic activity of a protein enzyme

Answer 14

the RNase P RNP where the catalytic RNA subunit’s activity is greatly enhanced by its protein cofactors which help position the tRNA substrate and stabilize the enzyme’s structure

Question 15

Provide a specific example of an RNA/RNP for how RNA-protein interactions can influence the catalytic activity of a ribozyme

Answer 15

bacterial RNase P which is essential for tRNA maturation where the protein component significantly enhances the catalytic activity of the ribozyme because while the RNA subunit alone can’t recognize and cleave tRNA, the addition of the protein dramatically increases the efficiency and expands the range of substrate RNAs it can act upon (protein does this by neutralizing the negative charges on RNA which improves substrate binding)

Question 16

What effect does phosphorylation have on the function of eukaryotic initiation factor 2
(eIF2)?

Answer 16

phosphorylation of eIF2 inhibits its function by preventing the exchange of GTP for GDP which is necessary for initiating protein synthesis

Question 17

Distinguish between the terms “knockdown” and “knockout” with respect to analyzing
gene function

Answer 17

Knockdown: temporarily reduces gene expression at the mRNA level resulting in partial and reversible loss of function
Knockout: permanently removes or inactivates a gene at the DNA level leading to complete and irreversible absense of the gene product

Question 18

Key steps in transgenic technology

Answer 18

insertion of transferred genetic material into the genome of an organism at a random site by pronuclear injection for gain of function analysis
DNA extraction (isolate DNA from organism and locate gene of interest in the extracted DNA), gene cloning (create multiple copies of gene of interest and link it with other DNA sequences to control its expression), transformation (introduce the gene with a vector to deliver it to the cells of the host and ensure it integrates into the host’s genome), and selection and breeding (select cells that successfully incorporated the new gene and grow into an organism)
Result: random integration of transgene

Question 19

Key steps in gene targeting

Answer 19

replacement or mutation of a particular gene in embryonic stem cells by homologous recombination; provides the means for creating strains of “knockout” organisms

engineer a targeting vector (design DNA construct that contains desired modification), introduce the targeting vector into embryonic stem cells/ES cells, select for and genotype the cells where the modification occurred, and generate a whole animal with the targeted gene (inject confirmed ES cells into mouse blastocyst)
Result: disruption or mutation of targeted gene
Recipient cell: Blastocyst stage embryo

Question 20

Key steps in gene editing

Answer 20

precise editing of targeted genome regions in virtually any cell type using the CRISPR-Cas system for loss/gain of function

Design a guide to a specific DNA target (pinpoint the sequence you want to alter and create a gRNA that will direct the editing machinery to the target), deliver the editing tools into cells (introduce editing complex into target cells), cut DNA at target site, leverage the cell’s natural repair mechanisms to delete or correct the gene (NHEJ or HDR if a DNA template is provided), then screen for successful edits
Result: precise editing of a specific gene (any cell type recipient)

Question 21

Key steps in cloning by nuclear transfer

Answer 21

a genetically-identical organism produced by nuclear transfer from adult somatic cells to an unfertilized egg to analyze genome reprogramming
Obtain a cell from animal to be cloned and a mature egg cell from donor, remove nucleus from donor, insert nucleus from animal cell in donor and fuse new nucleus with the egg and stimulate cell to start dividing, forms a blastocyst, implant blastocyst into surrogate
Result: genetically identical individual to donor nucleus
Recipient cell: enucleated egg cell

Question 22

A team of molecular biologists want to completely get rid of the expression of a particular gene in the cells they are working with in the lab. They tell you that they are designing a strategy using RNAi. Suggest a technique that could be more successful and explain your rationale.

Answer 22

Gene editing, CRISPR-Cas9 mediated gene knockout
It’s better because it’s a permanent loss of gene function, it can give a near complete knockout with a frameshift mutation yielding a stop codon (RNAi usually a partial knockout because some mRNA remains), it has high specificity (gRNA target exact genome sequences), and works on DNA (RNAi only affects transcripts already produced

Question 23

Consider the following hypothetical scenario:
A young scientific genius, Smarty, is terminally ill. When Smarty dies, Smarty’s parents
feel that one of the most remarkable minds in science will die with them, and the parents
feel they owe it to the world to not let this happen. The family travels to a secret lab on a
small offshore island which allegedly performs cloning by nuclear transfer. Smarty’s
parents hope to clone Smarty from one of Smarty’s skin cells.
Would Smarty and their clone have identical nuclear DNA profiles generated by DNA
typing? Explain your answer.

Answer 23

It would be almost entirely identical, but not perfectly
nuclear transfer involves taking the nucleus from his skin cell and placing it into an enucleated egg and because the clone receives his exact genome, the nuclear DNA profile would be the same
However, somatic mutations that occurred in the specific skin cell used for cloning would be passed to the clone so they could potentially differ at some loci due to mutations

Question 24

Consider the following hypothetical scenario:
A young scientific genius, Smarty, is terminally ill. When Smarty dies, Smarty’s parents
feel that one of the most remarkable minds in science will die with them, and the parents
feel they owe it to the world to not let this happen. The family travels to a secret lab on a
small offshore island which allegedly performs cloning by nuclear transfer. Smarty’s
parents hope to clone Smarty from one of Smarty’s skin cells.
Would Smarty and their clone share the same mitochondrial DNA? Explain your
answer

Answer 24

No, the egg cell used for cloning contains its own mitochondria and it’s inherited from the egg donor only, so the clone inherits mtDNA from the egg donor and not from Smarty

Question 25

Describe the underlying principles for DNA typing (DNA profiling/DNA fingerprinting) using short tandem repeat (STR) analysis and multiplex PCR

Answer 25

STR: short DNA sequences repeated, highly polymorphic because different individuals have different numbers of repeat units at each STR locus, this variation creates alleles of different lengths making STRs idea for distinguishing individuals... PCR: used to amplify STR regions from small DNA amounts, multiplex allows simultaneous amplification of multiple STR loci in a single reaction by having each locus use a distinct primer pair which is labeled with a distinct fluorescent dye... this increases efficiency, reduces sample consumption, and enables generation of a full DNA profile from small or degraded samples

Question 26

Genetic changes associated with cancer cells and tumorigenesis; distinguish between the following terms: driver mutation, oncogene, proto-oncogene, and tumor suppressor gene;

Answer 26

driver mutation: specific genetic alteration that provides a selective advantage for cancer growth, directly contributing to malignant phenotype Oncogene: mutated proto-oncogene that becomes hyperactive, causing continuous cell proliferation (stuck "on") proto-oncogene: normal gene that promotes cell growth, division, and survival tumor suppressor gene: normal gene that halts cell division or triggers cell death to prevent tumors

Question 27

Genetic changes associated with cancer cells and tumorigenesis; using retinoblastoma as an example, briefly explain Knudson’s “two-hit” hypothesis

Answer 27

hypothesis explains the difference between hereditary and sporadic forms of retinoblastoma... stating that two inactivating mutations must occur in both alleles of a tumor suppressor gene within a single cell for a tumor to develop Hereditary retinoblastoma... individuals inherit one defective allele through the germline and because they already have one hit, only a single spontaneous somatic mutation in the remaining healthy allele within a retinal cell is required to trigger cancer development Sporadic retinoblastoma... individuals born with two normal alleles and both the first and second mutations must occur spontaneously in the same somatic retinal cell during individuals lifetime for a tumor to form (low probability of occuring)

Question 28

Genetic changes associated with cancer cells and tumorigenesis; explain how deletions of both alleles of p53, or missense point mutations in one allele, can each lead to unrestrained cell growth

Answer 28

P53 is a tumor suppressor gene... if both alleles are deleted the cell produced no functional p53 protein causing the cell to be unable to sense DNA danage, activate repair pathways, and arrest the cell cycle leading to uncontrolled proliferation a missense mutation in one allele produces an abnormal p53 protein and even one mutant can disrupt the whole complex because mutant p53 mix with normal ones and make the entire tetramer nonfunctional so single missense mutation can inactivate p53 signaling almost as effectively as deleting both alleles

Question 29

is benzo(a)pyrene in cigarette smoke really a carcinogen?

Answer 29

potent carcinogen, a polycyclic aromatic hydrocaron that damages DNA by metabolizing and triggering mutations leading to lung, skin, and bladder cancers by disrupting cell growth and repair the body tries to break it down but this process creates a highly reactive intermediate which binds DNA and forms adducts which can cause permanent DNA mutations which activates genes that control cell growth potentially leading to uncontrolled cell growth

Question 30

Explain the terms “random walk,” “jumping,” and “induced fit” in the context of the mode of action of a restriction endonuclease

Answer 30

random walk: enzyme's non specific, rapid sliding along DNA to explore large regions quickly jumping: aka facilitated diffusion, rapid hopping between distant sites after the enzyme doesn't find its site, to speed up target search compared to just random walking induced fit: enzyme's flexible active site changing shape after binding the correct DNA site to precisely position the DNA for cutting

Question 31

Describe the principles underlying use of PCR-RFLP as a diagnostic test for a genetic disease

Answer 31

the detection of genetic variations (polymorphisms) in a specific DNA sequence based on whether these variations create or abolish recognition sites for specific restriction enzymes combines targeted DNA amplification with restriction enzyme digestion to reveal differences in fragment sizes between normal and mutant alleles amplify region containing mutation (PCR), exploit mutation's effect on restriction enzyme site, and compare fragment patterns on a gel to distinguish normal, mutant, and carrier genotypes

Question 32

Why are different gene therapy treatment strategies and different vectors used for different genetic diseases? Give specific examples.

Answer 32

each disease requires specific cell targeting, gene function, expression duration, and delivery method with vectors being chosen for their ability to infect certain cells or provide long-term vs transient gene expression SMA Zolgensma: AAV9 vector for in vivo delivery to motor neurons in the brain to replace missing SMN1 gene MLD: lentiviral vector to modify patients blood stem cells ex vivo with correct copy of the faulty gene then return to body to produce healthy cells CRISPR-based therapies: used ex vivo to precisely edit DNA in stem cells

Question 33

You are designing a somatic cell gene therapy protocol for ADA-SCID, a genetic disorder that is caused by a defect in adenosine deaminase. You have cloned the ADA cDNA. Name the minimal DNA regulatory element you would need to link to the ADA cDNA to get transcription of the cDNA in cells

Answer 33

a strong promoter... the core promoter, a DNA sequence containing the transcription start site and core elements that recruits the basal transcription machinery without proper promoter the polymerase won't initiate transcription of the cDNA

Question 34

Describe specific examples of how gene editing could be used or is being used to correct disease-associated mutations in humans

Answer 34

CRISPR: fixes faulty genes in cells (sickle cell disease)... restores healthy blood cells by editing stem cells outside the body (ex vivo) then reinfusing into the body resulting in patients producing healthy red blood cells and reducing symptoms... it can also inactivate disease genes (like disrupting HIV's entry) by T cells being editied to knock out the CCR5 gene (HIV gene) to prevent HIV from entering and infecting them (creates HIV resistant immune cells

Question 35

In a study that involved 100 college students, you find a polymorphism in a gene for a neurotransmitter receptor. The “long allele” is linked with reckless behavior. What conclusions, if any, can you draw about the results of this study? Should you hold a press conference to announce the discovery of the “reckless” gene?

Answer 35

You can't claim to have found a "reckless gene"... at best you have a preliminary association requiring replication so you would not hold a press conference to announce the discovery it shows preliminary association only, no proof of causation, and limited generalizability main limitations include small sample size, multiple testing risk, population stratification, measurement issues...etc.