Past Papers Flashcards
What is induced pluripotent stem cells and why are they inefficient to use them?
Induced pluripotent stem cells are a type of stem cells are generated by reprogramming adult cells into a pluripotent state. IPSC have remarkable ability to differentiate into any cell type found in the human body. Process: involves introducing specific genes, known as reprogramming factors, into adult cells. Such as OCT4: This gene plays a crucial role in maintaining pluripotency and self-renewal in embryonic stem cells. These reprogramming factors can activate or deactivate certain genes in the cells DNA, effectively reprogramming the cell and resetting it to an embryonic-like state. By doing so, we have pluripotent stem cells.
They are generally inefficient due to:
1. Epigenetic Barriers: Stable epigenetic marks in somatic cells resist reprogramming, making it difficult to revert them to a pluripotent state. 2. Variable Transcription Factor Expression: Inconsistent or insufficient expression of reprogramming factors (e.g., Oct4, Sox2, Klf4, c-Myc) leads to failed or incomplete reprogramming. 3. Cellular Senescence and Apoptosis: Cells may undergo senescence or programmed cell death due to the stress of reprogramming, reducing the number of successfully reprogrammed cells.
Describe how SMAD works. TGF-B pathway
SMAD proteins are used in TGF-B signaling pathway. Molecules that play a crucial role in transmitting signals from the cell membrane to nucleus. Brief explanation of TGF-B signaling pathway:
1- Ligand binding: When the ligand is available in the extracellular space, it binds to the receptor and two receptors get close to each other (dimerization) and R2 phosphorylates R1. After phosphorylation receptor type-1’s binding site become unmasked.
2- Activated R1 then phosphorylates specific SMAD proteins known as R-SMAD
3- R-SMADs find a partner in the cytoplasm called Co-SMAD and together they migrate to the nucleus.
4- Bind DNA directly and to promote the transcription of the target gene.
5- Anti-proliferation, apoptosis, cell cycle arrest happen.
List and describe the components of PCR
PCR is widely used technique that amplifies specific DNA sequences. Here is the list of components
of PCR:
1- DNA template: which contains the region of interest
2- Primers: short DNA sequences that are designed to bind to the regions flanking target DNA
sequences.
3- DNA Polymerase: Enzyme responsible for synthesizing new DNA strands from the given DNA
strands or probes.
4- Nucleotides(dNTPs): building blocks of DNA that are required for synthesis of the new strands
5- Buffer solution: provides necessary pH and ionic conditions for the reaction
6- MgCl2: an essential cofactor for DNA Polymerase activity
7- Thermal cycler: laboratory instrument that carries out the temperature cycling required for
PCR.
What is pre-microRNA
Refers to the precursor form of a microRNA molecule. MicroRNAs play an important role in gene
regulation. They are involved in the post-transcriptional regulation by binding to mRNA molecules, leading to their degradation or inhibition of translation. (22 nts in length). Pre-miRNAs are longer RNA molecules that undergo processing steps to generate mature miRNAs. Here is the steps:
1- pri-miRNA is transcribed by enzyme RNAP2.
2- Drosha processing: In the nucleus the pri-miRNAs recognized and cleaved by an enzyme called Drosha to generate hairpin shaped miRNA molecules known as pre-miRNA (70-100).
3- The pre-miRNA then exported into the cytoplasm.
4- Dicer processing: In the cytoplasm, a cleave enzyme called DICER cleaves the pre-miRNA,
removing the loop region, and generates a short RNA duplex with double strand.
5- Strand selection: One of the strands of RNA duplex is preferentially selected as mature miRNA
while other strand degraded.
6- Loading into RNA-Induced Silencing complex (RISC): The mature miRNA is incorporated into a
multi protein complex called RISC and miRNA guides RISC to bind complementary sequences on the target mRNA molecules.
7- Once bound to its target mRNA, miRNA can induce mRNA degradation or inhibit translation.
Deletion of the donor site
Refers to the removal or alteration of the sequence that serves as the donor site during a process
such as splicing or recombination. In context of splicing the donor site typically found in the 5’ end of an intron that helps in its recognition and removal during the splicing of pre-mRNA. Deletion of the donor site can lead to alteration in the splicing process thus can cause altered or non-functional protein. In recombination, the donor site is the region from which DNA segments are transferred or exchanged. Deletion of the donor site can disrupt the recombination process affecting the exchange of genetic material.
Explain the LAC operon agents such as promoter, operator, and terminator and how the operon works
LAC operon is the well-known example of gene regulation in prokaryotes. It is a negative inducible operon. LAC operon modulates the Lactose metabolism. Here is some agents of LAC operon:
1- Promoter: A DNA sequence located upstream of the genes in the operon. It serves as a
binding site for RNAP, the enzyme responsible for initiating transcription.
2- Operator: Is a DNA sequence situated between the promoter and structural genes of the operon. It acts as a regulatory switch, controlling gene expression. It serves as a binding site for a protein called the lac repressor. When the lac repressor binds to the operator, it physically blocks RNA polymerase from transcribing the structural genes, leading to the repression of the genes.
3- Terminator: Is a DNA sequence located at the end of the structural genes in the operon. It acts as a signal for RNAP to stop transcription, ensuring that RNA polymerase releases from the DNA after transcribing the structural genes.
The regulation of LAC operon primarily controlled by lac repressor protein and the presence or absence of lactose. In the absence of lactose, the lac repressor binds to the operator thus inhibiting the transcription of the structural genes. Even if the lactose is present the lac repressor protein will bind to the operator region thus inhibiting the transcription because glucose is still present, and we do not need lactase enzyme. This can be called negative regulation of the lac operon because it kept the structural genes in an inhibited form in presence of glucose. On the contrary Positive regulation of lac operon means which gene expression is enhanced by a regulatory molecule in this case it is called cAMP. When glucose levels are high cAMP levels are low thus cannot enhance the activity transcription process. Then cAMP and CAP proteins forms a complex and bind to the operator region if the operon thus enhancing the transcription.
What is an Attenuator?
Is a regulatory element found in operons that controls gene expression by modulating the termination of the transcription. It is typically found in operons that involved in the biosynthesis of amino acids and other metabolites such as Trp operon. The attenuator functions as a molecular switch that can terminate transcription prematurely under specific conditions. It enables the cell to fine-tune the expression of the operon’s genes based on the availability of the molecule that the operon is responsible for producing. The attenuator consists of specific DNA sequence within the leader region of the operon mRNA, like two tryptophan residues in a 14 aa peptide which is very high for normal cells. This peptide works as a sensor of amount of tryptophan available in the cell. This mechanism works only in the prokaryotic cells because transcription and translation occur simultaneously only in prokaryotic cells. The attenuation mechanism involves the formation of alternative stem-loops structures within the attenuator sequence, depending on the availability of the metabolite. Attenuation if there is high level of tryptophan anti-termination if there is low level of tryptophan in the cells.
What is chromodomain?
It is a protein domain involved in binding to methylated lysine residues on histone proteins. It
recognizes specific methylated sites on histones and facilitates protein-protein interactions. Chromodomains play a crucial role in regulating chromatin structure, gene expression and other
chromatin-associated processes. They can interpret the histone code and contribute to the establishment of specific chromatin states. Proteins containing chromodomains have diverse function such as gene activation, repression, and chromatin remodeling.
What is HOX genes what they do?
They encode for transcription factors. During embryonic development, HOX genes are expressed in a spatially and temporally specific manner along the body axis. The expression of different Hox genes is associated with different body regions. Homeobox sequence is highly conserved in animals because these genes regulate the TFs (master of masters). A subset of homeobox genes is hox genes are involved in the correct positioning of body parts in an organism. Several Hox genes are found next to each other on a chromosome, so they are arranged in clusters. The order of the genes on the chromosome matches the expression patterns along the embryo, showing spatial linearity. The genes that are expressed more posteriorly suppress the activation of genes that are expressed more anteriorly.
What is sporulation?
Is a process by which certain bacteria transform into a dormant and highly resistant form called spore. It is a survival mechanism that occurs in response to unfavorable environmental conditions. During sporulation bacteria undergo some reactions such as asymmetric cell division, formation of a spore coat and dehydration of the spore. This process allows bacteria to protect its genetic material.
What is WNT signaling pathway?
A highly conserved signaling pathway what plays a crucial role in embryonic development, tissue homeostasis and cell proliferation. WNT proteins bind to cell surface receptors known as Frizzled (FZZ) receptors and co-receptors such as LRP. This binding leads to inhibition of a protein complex called the B-catenin destruction complex, which normally promotes the degradation of B-catenin. When the destruction complex is inhibited B-catenin accumulates in the cytoplasm and translocate into nucleus. In the nucleus B-catenin interacts with TCF (T-cell factor) which is an repressive TF. This leads to activation of target gene. Functions: embryonic development, tissue homeostasis, cell fate determination, controls stem cells renewal and differentiation and contributes to tissue repair and regeneration.
Sigma factor 32
Is a bacterial sigma factor that plays a role in the regulation of heat shock response. Sigma factors
are bacterial transcription factors. When the bacterium is exposed to high temperatures or other
stressful conditions the levels of sigma32 increase rapidly. Sigma32 factor is involved in the transcriptional activation of shock response genes. These genes encode for heat shock proteins
(HSPs) and chaperons which are responsible for protecting bacteria from environmental stress.
Abortive initiation in eukaryotes/prokaryotes
Refers to the premature termination of transcription during the initiation phase. In prokaryotes, abortive initiation caused RNA polymerase stalling because sigma factor blocks the exit site of RNA polymerase so synthesized mRNA cannot go out of the RNA polymerase. RNA polymerase makes some unsuccessful attempts to initiate transcription. It repeatedly synthesizes and release short RNA fragments (2-10 nts). Abortive initiation in prokaryotes is believed to be a result of inefficient promoter clearance or inefficient transition from the closed complex to open complex.
In eukaryotes, RNAP synthesizes a short RNA fragment before transitioning into the elongation phase to produce full-length RNA transcript. Eukaryotic abortive initiation serves regulatory functions such as promoter clearance, facilitating the recruitment and positioning of transcription factors and co-regulators, and establishing transcription complexes for proper gene regulation.
The role of histone tail methylation in regulation of gene expression?
Methylation of histone tails primarily occurs at lysine (K) residues. It can activate transcription like H3K4Me this methylation allows for the recruitment of transcriptional activators and facilitating gene expression. H3K27Me inactivates transcription so it is a transcriptional inactivation. Also histone tail modifications can contribute to the epigenetic memory. Methylation of histone tails can affect the structure and accessibility of chromatin.
TBP unique feature?
TATA binding protein also called TBP is considered a TF. TBP recognize and bind to TATA BOX in the promoter region of the DNA. TBP is part of the pre-initiation complex in all promoters. TBP binds to the minor groove of the double helix which is very unusual. TBP bends DNA. TBP is also a core subunit of TFIID complex which is one of the basal transcription factors. Basal TFs provide a foundational machinery for gene transcription by ensuring the proper positioning and assembly of transcriptional machinery at the promoter.
Examples of default transcriptional repression and how it works?
It refers to suppression of gene expression in the absence of specific regulatory signals or factors. One of the factors could be Histone Deacetylation, histone tail acetylation generally associated with activation of gene expression. DNA methylation involves addition of methyl residues to cytosine residues within CpG dinucleotides. Polycomb proteins are a family of transcriptional repressor involved in development and maintenance of cell identity. They act by modifying chromatin structure and maintaining gene repression throughout cellular division. Also certain transcription factors function as repressor by directly binding to the promoter region within DNA.
SHH signaling pathway?
Sonic hedgehog signaling pathway, two receptors, Patched (PTCH) and Smoothened (SMO)
1- In the absence of hedgehog ligands, PTCH inhibits SMO, preventing its action. The pathway is inactive.
2- In the presence of hedgehog ligands, PTCH releases SMO and SMO becomes active. The pathway is active.
3- Activated SMO triggers a series of events, leading to activation of TFs called Gli proteins.
4- Activated Gli proteins translocate to the nucleus and regulate expression of genes. This genes are particularly important for cell proliferation, differentation and tissue patterning.
iPSC with knock-out of Dnmt3a and Dnmt3b, what to expect in their reprogramming?
They are two important methyltransferase enzymes several outcomes can be expected but we are going to talk about only one of them about cell reprogramming: Dnmt3a and Dnmt3b play roles in epigenetic reprogramming of somatic cells into iPSC. Their absence could hinder the reprogramming process and reduce the efficiency of iPSC generation. The altered DNA methylation patterns resulting from the loss of these enzyme might affect the ability of cells to undergo the necessary reprogramming events and acquire pluripotency. Removal of methyltransferase enzyme alone from a cell would not result in the generation of iPSCs. DNA methylation is an essential component of reprogramming. To generate iPSCs we need specific transcription factors like OCT4, Sox2, Klf4 and c-Myc.
TRP Operon regulation mechanism?
A system found in bacteria that regulates the production of tryptophan. TRP operon primarily
regulated by attenuation. Here is the steps:
1- Leader peptide and attenuator region upstream from the genes contains to trp residues within 14 aa peptide which is very high ratio in normal cells. The regulation of TRP operon is based on the availability of tryptophan in the cell.
2- The attenuation mechanism: In bacterial cells transcription and translation events are
concurrent. So when a mRNA synthesizing also translation happens. If the cell can find 2 trp
aa in a small amount of time loops 1-2 and 3-4 happens and attenuation happens.
3- Antitermination: If tRNAs cannot found trp aa easily within the cell then ribosome wait for
tRNA proteins in leader region thus region 1-2 cannot pair because of ribosome. Regions 2-3
form a loop called anti-termination loop. After tRNAs find trp and added it to the peptide ribosome go on and synthesize the needed peptide. 2-3 loop cannot block the ribosome while 3-4 loop can because 3-4 loop have more stable stem-loop structure formed by C-G base pairing regions between 3-4.
