Old exam 1 questions Flashcards
In E. coli DNA replication, the ring-shaped hexameric DnaB helicase travels along the single-stranded DNA in the 5’ to 3’ direction and unwinds double-stranded DNA at the replication fork as it migrates. In a standard replication fork, the strand of DNA that DnaB travels on serves as a template for the synthesis of what nascent molecule?
A- The leading strand of DNA
B- The lagging strand of DNA
C- Either strand of DNA
D- A strand of mRNA
E- There is not enough information
B the lagging strand
Draw a cartoon schematic of the replication fork with DnaB at work to illustrate your point (that DnaB is traveling on the template strand for lagging strand), marking the directionality of the parental and nascent DNA strands.
5’ —————-O———3’
3’ <–<–<–<– |
5’ ————–>|
3’ —————————5’
O = DNA B, on the template strand for synthesis of the lagging strand
Why do you think the prime editing system is less constrained by PAM than conventional CRISPR/Cas gene editing systems?
In conventional CRISPR editing, the position of the edit is dictated by the cut site (which takes place 3-4 nt upstream of PAM in the Streptococcus pyogenes system). In prime editing, the position of the edit with respect to PAM is dictated by the RT template of pegRNA (that gets copied by the RT (fused to Cas9 nickase) starting at the nicked site and extending for the entire length of the RT template). pegRNA can harbor a fairly long RT template (30nt+), with an edit in the nicked DNA introduced anywhere between the nick site and the 5’ end of the RT template (i.e. immediately after the nick site, in the PAM itself or significantly downstream of PAM, as far as the RT template allows).
Why do you think the human telomerase exists as a dimer, with two TERC and two TERT subunits present?
The two TERC/TERT units of telomerase work in parallel on the ends of two sister chromatids to ensure equal telomere extension.
In the 1958 Messelson-Stahl DNA replication experiment discussed in class (where E. coli grown in heavy 15N was moved to a light 14N source and a shift in the density gradient centrifugation pattern of genomic DNA was examined over time), what percentages of light, intermediate and/or heavy DNA do you expect to see after 2 hours (6 generations) of cell division in 14N? Assume that E. coli divides every 20 min.
0% heavy, 3% intermediate, 97% light
List four molecular technologies discussed in class that rely on emulsion PCR.
454 pyrosequencing
SOLiD sequencing by ligation
Ion Torrent sequencing by synthesis
Digital droplet PCR (ddPCR)
The copy number of DNA Pol III in E. coli is estimated at 10-20 copies of this multi-protein complex per cell. Why do you think the cell needs more copies of this essential protein complex than there are replication forks on its chromosome?
To participate in plasmid replication and in DNA repair.
A generic name of an enzyme that fuses the 3’-OH end of one DNA molecule to the 5’-P of another DNA molecule
DNA ligase
A generic name of an enzyme that removes the 5’-P on the molecule of DNA
phosphatase
A hybridization-based molecular method covered in class to study RNA distribution with cellular resolution in a slice of tissue
RNA in-situ hybridization
A fluorescence-based protein-protein interaction method that takes advantage of non-radiative energy transfer between two fluorescent proteins
FRET
A bacterial enzyme involved in nick translation
DNA Pol I
A eukaryotic enzyme that removes a stretch of single-stranded RNA/DNA displaced by DNA polymerase delta prior to Okazaki fragment joining
FEN1
A next-gen sequencing method that detects protons being released during DNA synthesis
ion torrent
Two kinases that phosphorylate eukaryotic replication licensing factors
S-phase CDK and Ddk
A generic name of a marker gene that assists in the identification or propagation of plasmids in bacterial or fungal strains with specific nutrient requirements
auxotrophic marker
A modern sequencing technology that enables the detection of specific chemical modifications in DNA or RNA
oxford nanopore or pacbio SMRT
A hexameric protein complex in eukaryotes that marks the origin of replication throughout the cell cycle
ORC1-6
A molecular method that determines the coordinates of a DNA-binding protein on its target DNA
DNase I footprinting (bp resolution level) or ChIP-seq/DAP-seq (maps binding to a 200-500bp region
An outdated gene expression quantification method that can be used to map how far the ends of a transcript extend
RNase protection assay
A protein in eukaryotes that confers processivity to replicative DNA polymerases delta or epsilon.
PCNA
(Proliferating cell nuclear antigen)
A DNA secondary structure that forms in repetitive sequences harboring direct tandem repeats
Slipped structure
A bacterial restriction enzyme utilized in two different DNA editing technologies
Fok1
A medium/high-throughput gene expression analysis method covered in class that involves an affinity purification step
nanostrings
A ribonuclear protein complex in eukaryotes that builds telomeric ends
Telomerase
A chemical property of a DNA double helix that has the most contribution to its stability
Base stacking due to their hydrophobicity
Why do you think the oriC of one bacterial species may not work in another bacterial species? How will you experimentally test in vivo and/or in vitro that your explanation is mechanistically correct?
DNA elements within oriC could have diverged in evolution and may not be properly recognized by other species’ DnaA (and other factors like IHF). If that is the case, then protein-DNA interaction assays like EMSA between the A-box and DnaA from the same and divergent bacterial species will show a lack of binding with mismatched protein-DNA pair, but good binding with matching combinations. For these experiments, A-box-containing DNA fragments can be amplified and labelled by PCR, whereas the DnaA protein can be expressed from a recombinant DNA construct in a cell-free in vitro transcription-translation reaction (this is to avoid the possible toxicity of DnaA when expressed in bacteria).
An alternative explanation is that the spacing between the DNA elements is different in different species (e.g. due to a slightly different size and hence the footprint of DnaA) that ultimately prevents proper oligomerization between DnaA monomers. The footprint of DnaA on the oriC fragment can be examined by DNase I footprinting and the oligomerization can be tested by EMSA (a supershift is expected) or a biochemical technique that we have not discussed in class called gel filtration (that separates protein (or in this case, protein-DNA) complexes by size). Other protein-DNA binding techniques (e.g., yeast-1-hybrid) can be employed to survey DnaA/A-box binding compatibilities of matched and mismatched DNA-protein combinations. PCR-based site-directed mutagenesis (a technique that introduces desired mutations via the primer sequences) can be performed to change DnaA sequences or the spacing between the A-box elements to see the effect of these mutations in vitro or in yeast on DnaA binding from both bacterial sources.
Note that working with oriC mutants in the native chromosomal context is probably not realistic, as the lack of chromosome replication (that may result from sequence edits) means the bacterial cultures will fail to grow (and there won’t be any DNA to even confirm that the edits took place let alone study replication dynamics). If you want to study oriC mutants in bacteria, you can use an oriC-containing plasmid instead and select for the plasmid using an antibiotic resistance marker gene.
Screening for protein-protein interactions using yeast 2 hybrid is often problematic when using transcription factors of interest as baits. This is because transcription factors can transactivate the reporter even in the absence of any bait-pray interaction. Can you suggest two experimental workarounds that bypass this transactivation issue and enable you to test for interactions between a transcription factor of interest and its possible partner using yeast 2 hybrid?
- Swap the bait and the prey. Placing a transactivating transcription factor in the prey vector and the putative interactor in the bait vector solves the problem.
- Subdivide the transcription factor into individual overlapping domains and use those domains that do not transactivate as baits to screen against the prey.
- Fuse the prey to a strong repressor (rather than an activator) domain and screen for the shutdown of the reporter activity upon the successful bait-prey interaction (the repressor domain of the prey with override the transactivation ability of the interacting bait) using colorimetric and fluorescent markers or a URA3 gene in combination with a chemical called 5-FOA. 5-FOA is toxic to cells with active URA3, so the yeast cells will only survive if they repress URA3 activity upon bait-prey binding.
- Use a transcriptional activation system that is based on RNA polymerase III (that normally transcribes tRNAs and other housekeeping non-coding RNAs) rather than RNA polymerase II (that transcribes protein-coding genes). The assumption here is that the bait transcription factor of interest that transactivates RNA pol II promoters should not transactivate an RNA pol III promoter, so switching to a Pol III system solves the transactivation problem. The pol III system relies on a prey construct fused to a subunit of a general transcription factor TFIIIC that indirectly (via TFIIIB) recruits RNA Pol III and turns the transcription of a reporter gene on if recruited to the DNA via the prey-DBD interaction. The reporter is driven by the regulatory promoter elements recognized by TFIIIC and TFIIIB, enabling the activation of this system upon bait-DBD and prey-TFIIIC interaction.
Imagine that you just joined a new lab that studies flower development and your first task is to determine if the gene PRETTY1 is expressed in flower samples from 10 rare (not sequenced) tropical species. Assume that PRETTY1 homologs have been previously sequenced and characterized in several model organisms and shown to be important for petal shape. You do not know the exact sequence of PRETTY1 in any of these rare tropical species (and have no budget to sequence them), but you do have multiple sequences (and clones) of PRETTY1 from several model plant species and know that the sequences of PRETTY1 vary between different plants but are all related. How will you go about testing whether PRETTY1 homologs are expressed in the flowers of valuable tropical samples? Propose two experimental approaches you can use and clearly state any protocol modifications that would be required to detect PRETTY1 being expressed (Hint: mentally transpose yourself in time to the past and think how scientists used to approach these types of questions before next-gen sequencing was invented).
cDNA sequences from all available PRETTY1 gene homologs from different species can be computationally aligned to one another and the most conserved regions of the sequences determined. The conserved regions can be used for developing a hybridization probe for a Northern blot and for designing a pair of degenerate primers* that would recognize the PRETTY1 gene from all or most species (*degenerate primers are pools of primers with every possible nucleotide present in each non-conserved position of the primer sequence).
Northern blot: Because all PRETTY1 sequences are related, a labelled DNA probe from a model species’ PRETTY1 (amplified from model organism’s RNA by RT-PCR or from a cDNA clone by PCR in the presence of labeled nucleotides) can be hybridized to a Northern blot where RNAs from 10 tropical flower samples (and a control model species RNA) were resolved. By using a lower temperature and higher salt during the probe hybridization and washing steps (less stringent conditions), binding of a partially complementary probe will be enabled and can be detected by autoradiography.
RT-PCR: Reverse transcribe RNA of the 10 species of interest (plus from a model organism as a positive control) using oligo-dT and then employ degenerate primers against the most conserved regions of the PRETTY1 gene for the amplification of cDNA fragments from all 11 RNA samples. The RT-PCR products from each species can be Sanger sequenced, new internal (aka nested) gene-specific primers designed and used for gene-specific RT-qPCR on the 10 samples (and a positive control) to evaluate PRETTY1 expression across all tropical genotypes.
In-situ hybridization: Using gene-specific PRETTY1 primers developed for the RT-PCR approach above, amplify short cDNA fragments of each tropical PRETTY1 gene (and a model plant control) into a bacterial vector that harbors specific RNA polymerase promoters (e.g., T3 and T7) surrounding the cloning site (subcloning is not a requirement, as T3 and T7 promoter sequences can be introduced at the flanks of the linear PCR fragments via PCR). Generate DIG-labeled sense and antisense RNA probes by in vitro-transcribing the cDNA fragments using T3 and T7 RNA polymerases. Hybridize the probes (sense and antisense probes for 11 PRETTY1 genes, including the positive control) to the slices of embedded, permeabilized flowers for the 11 respective species and detect the PRETTY1 signal in 22 in situ hybridization reactions using commercial anti-DIG antibodies coupled with an enzyme (e.g. horseradish peroxidase). Run an enzymatic assay and detect the PRETTY1 signal by monitoring the enzyme activity. The sense PRETTY1 probe is used as a negative control for the antisense probe complementary to the PRETTY1 transcript of interest.
Imagine that your lab is characterizing a new immortal cell line derived from a dog melanoma. You are tasked with examining the length of the telomeres in these canine cells and with testing whether the expression of telomerase genes is upregulated. Assume that you have access to both a small sample of healthy skin tissue from that founder dog and an unlimited supply of the immortal cells. Propose two complementary strategies (A, B) for estimating the number or length of telomeric repeats (using the methods covered in class or their extensions/variations) and two strategies for measuring telomerase expression levels (C, D).
(A) A hybridization-based quantitative DNA FISH approach (Q-FISH) – use a DNA or, ideally, a PNA probe (peptide nucleic acid, https://molecularcytogenetics.biomedcentral.com/articles/10.1186/1755-8166-6-42 ) complementary to the telomeric repeat and tagged with a fluorescent dye to detect the telomeres in metaphase spreads using fluorescence microscopy. The brightness of the fluorescent label hybridizing to the chromosomal ends will reflect the number of the repeats. You can compare the fluorescence intensity between the healthy sample and the immortal cell line sample using imaging software packages such as Image J.
(B) A qPCR-based approach – design primers for the telomeric repeats and for a single-copy gene in the genome as a normalization control. Telomeric primers have multiple binding sites (and hence will produce a smear of fragments of different sizes) but will show different levels of amplification (and hence of SYBR green or TaqMan fluorescence) from genomic DNA obtained from healthy tissue versus the cancer cell line. The telomeric repeat amplification data need to be normalized against qPCR data obtained for a control genomic locus (present in the genome only once) to control for different DNA inputs between samples. The design of PCR primers is critical, with intentional mismatches introduced into primers to prevent self-annealing of the forward and reverse primers (aka primer-dimer formation). One such clever primer design is presented here: https://www-ncbi-nlm-nih-gov.prox.lib.ncsu.edu/pmc/articles/PMC115301/ (see fig 1).
Other approaches, e.g. Telomere Restriction Fragment Analysis (a Southern blot with genomic DNA enzymatically digested by restriction enzymes, typically a mix of 4-cutters that do not recognize the telomeric repeats but degrade the rest of chromosomal DNA, and with the surviving telomeres detected using radioactively or fluorescently labelled telomeric probes) are acceptable for parts A and B.
(C) Telomerase expression can be analyzed at the RNA level for all three components (TERC, TERT and Dyskerin), e.g. reverse transcription of total RNA into cDNA using oligo-dT followed by qRT-PCR or ddPCR using gene-specific primer pairs and SYBR Green chemistry (or TaqMan probes). Again, the fluorescence/expression levels detected with gene-specific telomerase primers in the healthy tissue and the immortal cell line need to be compared and normalized against that of a control housekeeping gene like Actin.
(D) TERT and Dyskerin proteins in the healthy tissue versus the cell line can be analyzed by separating boiled tissue lysates by SDS-PAGE and detecting TERT and Dyskerin by Western blots using commercial primary and secondary antibodies. (TERC is not translated and is not relevant here). Some regions of these proteins are very conserved, so human and mouse antibodies should work well for dog samples (see, for example, https://www.labome.com/product/LifeSpan-Biosciences/LS-C110511.html) in combination with anti-human and anti-mouse secondary antibodies. To control for sample loading and allow for data normalization, an anti-ACTIN (or another housekeeping protein) primary antibody should be used in parallel. Secondary antibodies are conjugated to an enzyme or a fluorescent dye, enabling histochemical or fluorescent detection of TERT and Dyskerin.
Other methods, e.g., immunolocalization of TERT and Dyskerin or Northern blots of TERC, TERT and Dyskerin, are also acceptable gene expression evaluation techniques for parts C and D. Microarrays and RNA-seq are a poor choice for detecting the expression of just three genes. Various techniques based on TRAP (Telomerase Repeated Amplification Protocol) are also not a good choice for this question, as you were specifically asked to measure the expression rather than the activity of telomerase.
A form of naturally found dsDNA that is NOT right-handed
Z-DNA
A eukaryotic endonucleolytic enzyme that removes an RNA primer after nick-translation by the replicative DNA polymerase delta
FEN1 (+ DNA2)
A generic name of a molecular method to study protein-protein interactions in vivo by the reconstitution of fluorescent proteins separated into two halves; does NOT leverage yeast
BiFC
An essential protein complex in eukaryotic DNA replication that abbreviates the subunit names to their gene numbers in Japanese
GINS
A scientific discovery of certain nucleotide ratios in dsDNA that allowed Watson and Crick to infer specific nucleotide base-pairing
Chargaff’s rule
An enzymatic activity of DNA polymerases that increases the fidelity of DNA synthesis (be sure to indicate its directionality!)
3’-5’ exonuclease
A generic name of an enzyme that synthesizes short stretches of RNA in DNA replication
Primase
An alternative secondary structure in DNA that is the culprit of repeat expansions in several human neurodegenerative disorders and muscular myopathies
Slipped structure
A version of Cas9 that has one of its nuclease domains disabled via a mutation
Nickase (D10A)
An amplification-based method of quantifying gene expression that relies on a displacement and degradation of an internal labelled probe during DNA synthesis
TaqMan RT-qPCR
A DNA-binding bacterial protein that can block the progression of a replication fork if it is approached from its non-permissive side
Tus
A method of interrogating interactions between a protein of interest and a piece of labelled DNA on a polyacrylamide gel; does NOT require the addition of any enzymes
EMSA
A sequence property of all prokaryotic and eukaryotic origins of replication
AT-rich
A protein-denaturing detergent utilized in most plasmid DNA extraction protocols
SDS
A phenomenon of transmitting energy between two juxtaposed reporter proteins
FRET
A display of hundreds of tiny droplets of different recombinant proteins on a solid surface
Functional protein microarray
