Preguntas de clase Flashcards
Assuming that there were no time constraints on replication of the genome of a human cell, what would be the minimum number of origins that would be required?
If there were no time constraints on replication, one origin would be required for each chromosome; thus, a minimum of 46 origins, equal to the number of chromosomes in a human cell, would be needed.
What consequences can you think of that would arise if a eukaryotic chromosome (150 Mb in length) had one of the following features:
* A single replication origin located in the middle of the chromosome (DNA replication proceeds at about 150 nt pairs per second).
* A chromosome with no telomeres
* No centromere
A single replication origin in the middle of the chromosome: Slow Replication, limit severily the cell division
A chromosome with no telomeres: would loose nucleotides during each DNA replication, essential genes would be lost
No centromere: crucial for proper chromosome segregation during cell division. Without a centromere, chromosomes might not be correctly distributed to daughter cells during mitosis and meiosis.
(T/F) Human cells do not contain any circular DNA molecules
False. some mitochondrial DNA molecules are circular
Why is Hoechst used for in vivo imaging ,…. but DAPI rarely used , and PI not used
Hoechst: bind specifically to the minor groove of DNA, resulting in strong fluorescence signals within the cell nuclei. This makes them useful for labeling and visualizing the nuclei in living cells.
DAPI: excessive exposure to UV light during live-cell imaging can lead to cell damage. This phototoxicity can limit the use of DAPI in prolonged in vivo imaging experiments.
Propidium Iodide (PI): is excluded from live cells with intact plasma membranes, and it only enters cells with compromised membrane integrity, such as dead or apoptotic cells. As a result, PI is not suitable for in vivo imaging of live cells.
Can you think of disadvantages of using UV light to visualize DNA/RNA?
DNA and cellular damage: phototoxic, mutations and apoptosis
The Human genome project and the company Celera used 2 different approaches to fully sequence the human genome.
Sequencing of mapped clones (=Bacterial Artificial Chromosomes) vs. shotgun sequencing
Shotgun sequencing is much faster compared to traditional methods but offers very short read length. If you think about the composition of DNA sequences in the human genome, what are putative problems a shotgun approach could face?
Challenges with Repeat Regions: The human genome contains a significant portion of repetitive elements, such as transposons and tandem repeats. Short read lengths in shotgun sequencing may make it difficult to resolve and assemble these repetitive regions accurately, leading to gaps or misassemblies in the genome
Difficulty in Resolving Structural Variations: Structural variations, including insertions, deletions, duplications, and inversions, are prevalent in the human genome. Short reads from shotgun sequencing may not provide enough information to accurately resolve complex structural variations, leading to challenges in reconstructing the true genomic architecture.
Which mutational changes would you predict to be the most deleterious to gene function?
Removal of a single nucleotide near the beginning of the coding sequence is noted as harmful. This is because it is likely to cause a frameshift mutation, altering the reading frame and leading to the production of a completely different protein or a truncated version.
There is a persisting expression of AAV transgene for years in a patient with retinal dystrophy. However, after 5-6 years there is a loss of expression. What are the possible reasons for that?
1) The patient’s immune cells might recognise the AAV vector at some point and eliminate it.
2) Epigenic modifications may silence the transgene / its promotor
3) Cells in the retina have a limited lifespan and eventually are replaced by new cells, lacking the AAV
Integrating viral vectors can lead to genotoxic events e.g., inflammation, random insertion disrupting normal genes, activation of proto-oncogenes, and insertional mutagenesis. Explain possible genotoxicity effects
A (i)
Promotor insertion upstream of our transcription unit -> expression of both transgene and cellular gene -> may lead to toxicity because of many copies of the protein present
A (ii)
Also insertion in the promotor region of the cellular gene may also result in genotoxicity due to the disruption of the original promotor and the possibly altered gene activation by the transgene LTR.
B (i)
Insertion of transgene cassette upstream of a cellular expression cassette may result in an overexpression of the cellular gene through transgene promotor / LTR activation
(e.g. if the cellular gene is a proto-oncogene, its increased expression might result in cancer-development)
B (ii)
Downstream activation of the cellular gene through LTR / promotor activation can lead to overexpression of the cellular gene, which often results in genotoxicity.
C
Intragenic insertion may lead to disruption of the mRNA and thus lead to a truncated mRNA. Therefore the gene may be inactivated.
Briefly explain 1 repair mechanism of single stand break and 1 repair mechanism of a double strand break.
Single strand:
-Baseexchange
-Nucleotide excision repair: scans DNA for distorsion in double helix-> cleaves the phosphodiester backbone -> DNA -> DNA helicase removes the sequence -> DNA polymerase and DNA ligase binds again a new sequence
Double strand:
-Homologous repair: newly replicated DNA, use of the sister chromatide as template, takes place once the DNA is repliccated for cell division
-Non homologous end joining: broken ends are brought together by ligases
What happens if the cell doesn’t have a centromere?
The primary role of the centromere is to facilitate the accurate separation of chromosomes during cell division. Without a centromere or with a defective centromeric region, chromosomes may not properly align and segregate during mitosis or meiosis.
What are the key differences between prokaryotes and eukaryotes in protein synthesis ?
Prokaryotes: Lack membrane-bound organelles, including a nucleus. Eukaryotes: Transcription occurs in the nucleus, and the primary transcript (pre-mRNA) undergoes processing, including splicing and capping, before it is transported to the cytoplasm for translation.
What is genotoxicity?
substance or agent to cause damage to genetic information-> genetic mutations, chromosomal aberrations, or other genetic alterations. including the development of cancer.
Which repair mechanisms do you know in eukaryotes?
Single strand:
-Nucleotide excision repair: scans DNA for distorsion in double helix-> cleaves the phosphodiester backbone -> DNA -> DNA helicase removes the sequence -> DNA polymerase and DNA ligase binds again a new sequence
Double strand:
-Homologous repair: newly replicated DNA, use of the sister chromatide as template, takes place once the DNA is repliccated for cell division
-Non homologous end joining: broken ends are brought together by ligases
How does the end mammalian dna sequence looks like?
Telomeres
What happens if the cell doesn’t have a telomere?
protect the ends of chromosomes from degradation and prevent them from sticking together. In the absence chromosomes become unstable. When telomeres become critically short, they can trigger a state of cellular senescence. If telomeres become extremely short, the cell may undergo apoptosis
How are viral vectors used in gene expression and name a genetic disease which can be treated?
AAVs are used to deliver therapeutic genes to target tissues, and they are particularly effective for treating diseases. First there is the genome design, then the transgene is cloned in the adenosin associated virus -> cell culture cells for AVV production -> transfection of cells. pathogenic, single-stranded DNA parvovirus. Recombinant AAV (rAAV) vectors’ excellent safety profile and durable gene expression have made it the preferred vector for gene therapy in the nervous system.
Disease: spinal muscular athrophy
Name the key processes in protein production and which steps influence the concentration of the produced protein?
Transcription-> capping, splicing, and polyadenylation-> Mature mRNA is transported out of the nucleus to the cytoplasm-> Translation -> Post-Translational Modifications -> protein folding
The concentration of the produced protein depends on each step
What is the difference between housekeeping genes and regulated genes?
Housekeeping are expressed all the time, regulated genes are controlled for differentiation
Can you think of one feature that make miRNAs especially useful regulators of gene expression?
ability to target multiple genes, used for gene silencing and inhibition
What is the difference between homodimer and heterodimer?
homodimer is a protein made from two identical proteins, while heterodimer is a protein made from two different proteins.
Many transcription regulators form dimers of identical or slightly different subunits on the DNA. Suggest advantages of dimerization.
Increased DNA Binding Specificity, Enhanced Stability on DNA, Cooperative Binding, Functional Diversity
Consider the argument:
If expression of every gene depends on a set of transcription factors, then the expression of these TF must also depend on the expression of other TF regulating their expression, which are in turn again regulated by a set of TF……..
Why does the cellular genome not code for an infinite number of transcription factors?
Combinatorial control is a common strategy in transcriptional regulation, where multiple transcription factors work together to regulate the expression of a gene. This allows for a diverse range of gene expression outcomes without requiring an individual transcription factor for every possible regulation.
Designing an expression casette:
* What sequence elements are present in the 5‘-UTR and 3‘-UTR elements of
delivered RNAs?
* Can you find out how these elements have been designed for clinical
applications or in laboratory experiments?
internal ribosome entry sites (IRES), and the 5’-cap structure enhance translation initiation and stability. The 3’-UTR includes elements such as polyadenylation signals, termination codons, and stability/localization elements crucial for mRNA stability, termination, and regulation.
For clinical applications or laboratory experiments, several design strategies are employed:
Codon Optimization: Optimizing codons for the host organism improves translation efficiency. Regulatory Elements: Inclusion of enhancers or repressors for modulating gene expression. Cell-Specific Promoters: Choosing promoters active in the target cell type ensures cell-specific expression. Vector Systems: Using viral (e.g., lentiviruses) or non-viral vectors (e.g., plasmids) for efficient delivery. Safety Features: Incorporating safety elements like insulators or inducible systems to enhance safety in clinical applications.
Can you think of differences between bacteria and animal cells that are, or could be, depended on the appearance or presence of the eukaryotic cytoskeleton?
Animal cells, with the support of the eukaryotic cytoskeleton, exhibit diverse shapes and can change their morphology. The cytoskeleton, consisting of actin filaments, microtubules, and intermediate filaments, provides structural support, maintains cell shape, and enables cell motility.
Bacterial cells often have a defined shape, which is maintained by the bacterial cytoskeleton
Bacterial cells lack membrane-bound organelles and a well-defined cytoplasmic structure.
The eukaryotic cytoskeleton, especially microtubules and actin filaments, contributes to the organization of organelles, intracellular transport, and the maintenance of cellular polarity.
The amino acid sequences of actin and tubulin in eukaryotic species are remarkably well conserved, while the numerous proteins that interact with the cytoskeleton are no more conserved than most other proteins.
How can it be that the filament proteins themselves are highly conserved, while the proteins that interact with them are not?
the high conservation of actin and tubulin in eukaryotic species reflects their central and essential roles in cellular structure and function. In contrast, the interacting proteins exhibit more sequence variability due to their diverse functions, evolutionary plasticity, and species-specific adaptations to meet the specific requirements of different organisms and cellular pathways.
How could a centrosome „sense“ when it is located in the „center“ of the cell?
The centrosome contains a pair of centrioles and serves as the main microtubule-organizing center (MTOC). Microtubules emanate from the centrosome and interact with various cellular structures. The dynamic nature of microtubules, influenced by polymerization and depolymerization, contributes to the positioning of the centrosome within the cell.
Kinesin-1 motos are highly processive, meaning they move along the tubulin tracks without dissociating.
In contrast, myosin II motors are not processive and dissociate after „a few steps“.
Think about their biological role, do these characteristics of the motor proteins make sense?
Kinesin-1:
Processivity: Highly processive, meaning it can move along microtubule tracks for long distances without dissociating.
Biological Role: Involved in anterograde transport, efficiently moving cellular cargoes, such as vesicles and organelles, along microtubule tracks to specific destinations within the cell.
Myosin II:
Lack of Processivity: Not highly processive and tends to dissociate after a few steps along actin filaments.
Biological Role: Involved in muscle contraction, cytokinesis, and other actin-based cellular activities. The lack of processivity allows myosin II to maintain control over force generation and localized interactions with actin filaments.
The distinct characteristics of kinesin-1 and myosin II motors are well-suited for their respective biological roles, reflecting the adaptability of motor proteins in facilitating diverse cellular functions with precision.
What are the functions of 3 different types of filaments
Intermediate filaments: * mechanical strength (large deformation without failure)
Microtubules: * determine positions for membrane enclosed organelles
* direct intracellular transport; cilia and flagella
* formation of mitotic spindle (chromosome segregation)
Actin filaments: * shape of a cells surface
* whole-cell locomotion
What is RNA editing?
alteration of RNA sequences after transcription, leading to changes in the information encoded by the DNA. Deamination, insertion/deletion
What are artificial modifications of mRNA used in medicine and transfection?
- Nucleotide methylation: increase stability and protein translation and decreases degradation and immunogenicity
- Codon optimization increases mRNA abundance and translation
- Nucleoside modification: increase stability, decrease degradation and immunogenicity
- Tail elongation: increase stability and protein translation
Cells are able to remodel the chromatin to make transcription happen. How do the cells manage this?
- Direct local alterations in chromatin structurethroug the reader writer complex: increaseaccessibility of DNA, facilitate binding of RNA pol and transcription factors. Nucleosome remodeling, nucleosome removal, histone replacement and certain types of histone modifications favor transcription.
Mechanical stress induces depolymerization of tubulin or of actin?
Actin
How cells sense?
This suggests that actin microfilaments are sensors of mechanical stretch in cells, and can form a feedback loop to control the mechanical tension of tissues.
Mzosinrings can sense small nanoscale structures
Actin associated motor proteins and functions in cells?
Motor proteins: movement of cell
myosin I -> all cells -> pull it to different shape
myosin II -> muscle cells:
- tail-tail interactions
- formation of bipolar
„thick filaments“
contraction of each myofibril (bundle of sarcomeres)
Keratin functions in cells and advantages of it as a biomaterial
Keratins: most diverse intermediate filament family
mechanical strength and integrity of tissues such as skin, hair, and nails. Its functions include providing structural support, maintaining epithelial tissue integrity, influencing cellular shape and rigidity, facilitating cellular adhesion,
can be used for the formation of hydrogel, microcapsules, sponges, nanofibers and films and is used in TE for wound healing, bone and cornea regeneration and drug delivery.
have an intrinsic ability to self-assemble and polymerize into porous, fibrous scaffolds
Explain Riboswitch
short sequences of RNA that change their conformation when bound to small molecules, like metabolites-> regulate gene expression. blocking or permitting progress of RNA polymerase. Eg. A riboswitch that responds to guanine. (A) In bacteria, the riboswitch controls expression of the purine biosynthetic genes. When guanine levels are low, an elongating RNA polymerase transcribes the purine biosynthetic genes, and the enzymes needed for guanine synthesis are therefore expressed. (B) When guanine is abundant, it binds the riboswitch, causing it to undergo a conformational change that forces the RNA polymerase to terminate transcription.
Name chemical Histone modifications and how they are applied and removed?
-acetylation can stimulate transcription initiation and the repressor simply reverses this modification
- methzlation:methylated histones are bound by proteins that maintain the chromatin in a transcriptionally silent form.
Mechanical stress induces depolymerization of …
Actin
Explain Actin Hydrogels and what can be researched with
Actin plays a role in muscle contraction, cellular movement. actin polymerization dynamics in nature vs. active gels, study on the formation of contractile fibers. Actin hydrogels are three-dimensional networks formed by the polymerization of actin monomers. Actin hydrogels allow the investigation of cellular mechanics, adhesion, and responses to mechanical cues.
Artifical phospholipid bilayer vesicle formed from an aqueous suspension of phospholipid molecules?
Liposome
