RNA Interference and Gene Silencing

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Questions and Answers

What is the primary function of restriction endonucleases in bacteria?

To assist in protein synthesis

To facilitate DNA replication

To synthesize RNA from DNA

To provide defense against foreign DNA (correct)

What is one of the technologies that has minimized the need for restriction endonucleases?

Sanger sequencing

Alternative cloning technologies (correct)

Gene transfer techniques

Polymerase Chain Reaction (PCR)

Which sequence does EcoRI recognize?

GAATTC (correct)

AAGCTT

CCGGAATTCCG

NNNGAATTCNNN

How many distinct recognition sites do bacteria typically have for restriction endonucleases?

More than 100 Signup and view all the answers

What is a potential application of restriction endonucleases in genetic engineering?

Cloning DNA fragments Signup and view all the answers

What is the main purpose of antibiotic resistance genes in plasmid vectors?

To enable selection of bacteria carrying the plasmid Signup and view all the answers

Which type of vector can accommodate the largest DNA inserts?

Yeast Artificial Chromosomes (YACs) Signup and view all the answers

What function does reverse transcriptase serve in RNA cloning?

It converts RNA into DNA Signup and view all the answers

Which of the following vectors is specifically designed for high levels of gene expression?

Expression Vectors Signup and view all the answers

What can be explored by isolating mRNA as a molecular clone?

Noncoding sequences in eukaryotic genes (introns) Signup and view all the answers

What is RNA interference (RNAi) primarily used for?

Gene specific silencing posttranscriptionally Signup and view all the answers

What type of RNA is required for the action of siRNA?

Double-stranded RNA approximately 21-22 nucleotides long Signup and view all the answers

What is one of the effects of aberrant miRNA expression?

Correlation with cancers Signup and view all the answers

How does siRNA exert its effect on target mRNAs?

By activating endonucleases that digest target mRNA Signup and view all the answers

What is the role of the RNA-induced silencing complex (RISC) in miRNA function?

To incorporate the miRNA strand for target mRNA interaction Signup and view all the answers

What advantage does siRNA have over conventional antisense RNAs?

siRNA works in a catalytic manner and at very low concentrations Signup and view all the answers

What are the consequences of posttranscriptional inhibition in gene regulation?

It can lead to potent antiviral responses. Signup and view all the answers

What was the approximate cost to complete the Human Genome Project?

$2.7 billion Signup and view all the answers

What significant advancement does 3rd generation sequencing offer compared to earlier methods?

Single molecule sequencing Signup and view all the answers

Which of the following represents the percentage of the genome that the Whole Exome Sequencing targets?

2-3% Signup and view all the answers

Which sequencing method is noted for being able to sequence the human genome in about 1 hour?

3rd generation sequencing Signup and view all the answers

What is one of the main applications of next generation sequencing?

Whole exome sequencing Signup and view all the answers

What is the primary function of the 16S rRNA gene in prokaryotic organisms?

To serve as a structural component of ribosomes Signup and view all the answers

How can the 16S rRNA gene be useful in studying bacterial evolution?

It has conserved and variable regions that indicate evolutionary relationships Signup and view all the answers

What characterizes the variable regions of the 16S rRNA gene?

They contain specific sequences unique to each species Signup and view all the answers

In nucleic acid hybridization, what is the purpose of raising the temperature during the initial stage?

To separate DNA strands Signup and view all the answers

What is the role of labeled DNA in Southern blotting?

To serve as a probe for hybridization with target sequences Signup and view all the answers

What is the primary function of bacterial flagella?

Movement toward nutrients or away from toxins Signup and view all the answers

How does the capsule contribute to bacterial virulence?

Provides resistance to phagocytosis Signup and view all the answers

What key component stabilizes bacterial spores and protects DNA?

Dipicolinic acid Signup and view all the answers

Which of the following describes the nature of the capsule in bacteria?

Thick and viscous or thin and gummy Signup and view all the answers

What is the main reason spore-forming bacteria can survive harsh conditions?

Ability to enter a dormant state Signup and view all the answers

What is the primary component of the bacterial cell wall that gives it strength?

Peptidoglycan Signup and view all the answers

During the Gram staining process, what is the role of Gram's iodine?

To form a complex with crystal violet Signup and view all the answers

How does the Gram-negative bacterial cell wall differ from that of Gram-positive bacteria?

It contains lipopolysaccharides Signup and view all the answers

What effect does washing bacterial cells with alcohol during Gram staining typically have?

Dehydrates the cell wall and removes the crystal violet stain in Gram-negative cells Signup and view all the answers

What structures in bacteria can activate macrophages and stimulate cytokine production?

Peptidoglycan and lipoteichoic acid Signup and view all the answers

Which of the following correctly describes the function of lipoteichoic acid in Gram-positive bacteria?

Retains crystal violet during Gram staining Signup and view all the answers

Why might certain bacterial species have unique cell walls?

To resist phagocytosis Signup and view all the answers

What structural feature primarily distinguishes Gram-positive bacteria from Gram-negative bacteria?

Thickness of the cell wall Signup and view all the answers

What is a common mechanism of antibiotic resistance in bacteria related to cell wall synthesis?

Production of b-lactamase enzymes Signup and view all the answers

What is the primary function of peptidoglycan in bacterial cells?

Providing structural integrity Signup and view all the answers

Which component of the bacterial cell wall is structurally related to β-lactam antibiotics?

All of the above Signup and view all the answers

What is the primary mechanism of resistance to penicillin in Gram-positive bacteria?

altered penicillin binding proteins Signup and view all the answers

What type of β-lactamase inactivates most β-lactams except for carbapenems?

Extended-spectrum β-lactamases (ESBLs) Signup and view all the answers

How do β-lactamase enzymes affect antibiotic efficacy?

They hydrolyze β-lactam antibiotics, rendering them ineffective. Signup and view all the answers

Which type of bacteria are most commonly associated with β-lactamase enzyme resistance?

Both Gram-positive and Gram-negative bacteria Signup and view all the answers

What is the result of β-lactam antibiotics binding to the transpeptidase active site?

Promotion of autolysis Signup and view all the answers

What mechanism is utilized by MRSA to resist penicillin?

Decreased target site affinity Signup and view all the answers

Which of the following best describes the structure of β-lactamase enzymes?

Ribbon structures that are secreted Signup and view all the answers

What is one of the roles of porins in Gram-negative bacteria regarding antibiotic resistance?

Decrease antibiotic penetration Signup and view all the answers

Which component of the Gram-negative outer membrane is primarily responsible for triggering an immune response?

Lipopolysaccharide (LPS) Signup and view all the answers

What distinguishes the peptidoglycan layer in Gram-negative bacteria from Gram-positive bacteria?

It is sandwiched between two membranes in Gram-negative bacteria. Signup and view all the answers

What is the primary role of the core polysaccharide in the lipopolysaccharide molecule?

To provide structural integrity to LPS. Signup and view all the answers

Which of the following correctly describes the function of O-antigen in bacteria?

It is used for bacterial serotyping. Signup and view all the answers

Which structure allows for the passive diffusion of small, charged molecules in the outer membrane of Gram-negative bacteria?

Porins Signup and view all the answers

What is one of the key characteristics of the immune response triggered by lipopolysaccharides?

It activates cytokine release from macrophages. Signup and view all the answers

During septic shock, what is primarily attributed to the endotoxic activity of LPS?

Activation of complement and coagulation cascades. Signup and view all the answers

What role do Toll-Like Receptors (TLRs) play in the immune response to LPS?

They facilitate the binding of LPS and initiate a signaling cascade. Signup and view all the answers

What is the composition of the bacterial cell wall primarily based on?

Peptidoglycan cross-linked by peptide chains Signup and view all the answers

How does the Gram staining process differentiate between Gram-positive and Gram-negative bacteria?

By their ability to retain crystal violet after alcohol washing Signup and view all the answers

What role does lipoteichoic acid play in the structure of Gram-positive bacteria?

It contributes to structural integrity and may play a role in immune response Signup and view all the answers

Which factor contributes to the virulence of bacteria with unique cell walls?

Resistance to phagocytosis and immune evasion Signup and view all the answers

Which of the following statements regarding β-lactam antibiotics is true?

They bind to the transpeptidase enzyme, disrupting cell wall synthesis. Signup and view all the answers

What is the primary difference between Gram-positive and Gram-negative bacteria in terms of their cell envelope structure?

Gram-positive bacteria have a single plasma membrane, while Gram-negative have two membranes. Signup and view all the answers

What is the primary consequence of β-lactam antibiotics binding to the transpeptidase active site?

Inhibition of cell wall synthesis leading to autolysis Signup and view all the answers

What is the primary role of the outer membrane in Gram-negative bacteria?

Acts as a permeability barrier for large molecules Signup and view all the answers

Which mechanism of penicillin resistance involves alterations to penicillin-binding proteins?

Genetic mutations resulting in MRSA Signup and view all the answers

Which statement accurately reflects the structure of Gram-negative peptidoglycan?

It has less overall crosslinking resulting in increased porosity. Signup and view all the answers

Which type of β-lactamase is specifically designed to inactivate most β-lactams except for carbapenems?

Extended-spectrum β-lactamases (ESBLs) Signup and view all the answers

What is a characteristic of bacterial ribosomes that enables selective toxicity of certain antibiotics?

The presence of a 30S subunit that differs significantly in structure. Signup and view all the answers

Which of the following statements correctly describes the function of pili in bacteria?

They assist in adherence to host cells and other bacteria. Signup and view all the answers

In which bacterial type is β-lactamase primarily synthesized and secreted into the periplasm?

Gram-negative bacteria Signup and view all the answers

What is the result of reduced penetration of β-lactam antibiotics in Gram-negative bacteria?

Decreased effectiveness of β-lactam treatment Signup and view all the answers

Which property of the periplasmic space is significant for Gram-negative bacteria?

It harbors hydrolytic enzymes and components for sugar transport. Signup and view all the answers

What role do LPS molecules play in the outer membrane of Gram-negative bacteria?

They act as molecular signals to the host organism. Signup and view all the answers

Which component of the bacterial cell wall is structurally related to β-lactam antibiotics?

D-Ala-D-Ala peptide Signup and view all the answers

What is a significant characteristic of β-lactamase enzymes in Gram-negative bacteria?

They are mostly encoded on plasmids Signup and view all the answers

Which structural alteration is primarily responsible for the resistance of penicillin-resistant S. pneumoniae?

Altered penicillin-binding protein PBP2x Signup and view all the answers

What component of lipopolysaccharide (LPS) is primarily responsible for its endotoxic activity?

Lipid A Signup and view all the answers

Which characteristic of Gram-negative bacteria's outer membrane helps facilitate passive diffusion of small molecules?

Porins Signup and view all the answers

What property of lipopolysaccharides makes them poorly antigenic despite being strong immune stimuli?

Lack of structural variation Signup and view all the answers

Which part of lipopolysaccharide (LPS) is specifically composed of an unusual eight-carbon sugar?

Core polysaccharide Signup and view all the answers

Which of the following cytokines is NOT released by macrophages upon activation by lipopolysaccharide?

Interferon gamma Signup and view all the answers

What triggers the activation of macrophages by lipopolysaccharide (LPS)?

Toll-like receptor 4 (TLR4) Signup and view all the answers

Which feature of Gram-negative bacteria's cell envelope best explains their structural integrity?

Lipid bilayer of the outer membrane Signup and view all the answers

What consequence might occur due to the shedding of lipopolysaccharide (LPS) into the bloodstream during bacteremia?

Induction of septic shock Signup and view all the answers

Which statement accurately describes the composition of lipopolysaccharides?

LPS contains a lipid component and polysaccharides. Signup and view all the answers

What is the primary role of the core polysaccharide in lipopolysaccharide structure?

Essential for LPS structure and bacterial viability Signup and view all the answers

Study Notes

RNA Interference

RNAi refers to gene-specific silencing occurring post-transcriptionally.
Involves silencing RNA technologies like siRNA and miRNA.

Posttranscriptional Inhibition

A form of gene silencing inhibiting RNA after transcription.
Transcriptional gene silencing (TGS) faces challenges including RNA stability and antiviral responses in mammals.

MicroRNA (miRNA)

Highly conserved mechanism with 300-800 miRNA genes in humans.
Regulates expression of approximately 30% of all protein-coding genes.
Linked to cellular processes such as development, proliferation, metabolism, and differentiation.
Aberrant expression of miRNAs can correlate with cancer.
The final step in miRNA function is incorporation into the RNA-induced silencing complex (RISC).
Target sites for miRNAs are predominantly found in the 3' UTR of mRNAs.

Small Interfering RNA (siRNA)

siRNAs are 21-22 nt long and derived from double-stranded RNA.
Operate in a catalytic manner and require low concentrations to be effective.
More potent than conventional antisense RNAs.
Function by activating endonucleases that digest complementary mRNA.

Recombinant DNA Technologies

Utilize restriction endonucleases (REs) which cleave DNA at specific sequences, providing defense against foreign DNA in bacteria.
Multiple types of cloning vectors exist, including plasmids, cosmids, BACs, and YACs, each with functions suited for varied insert sizes.

Cloning and Vectors

Plasmids can clone DNA inserts up to a few thousand base pairs, featuring an origin of replication and antibiotic resistance genes.
Cosmids allow for DNA inserts up to 50 kb; BACs accommodate 150-300 kb; YACs handle 200-500 kb.
Expression vectors are designed for high levels of gene expression.

RNA Cloning

RNA is cloned by reverse transcription into cDNA integrated into vectors.
Enables exploration of non-coding sequences in genes (introns) and production of proteins requiring post-translational modifications.

DNA Sequencing

The Human Genome Project, completed in 2001, cost $2.7 billion and took 5 years.
Advances in sequencing technology reduced the time and cost of sequencing a human genome significantly, with third-generation sequencing achieving it in one hour for 100−100-100−1000.

Next-Generation Sequencing Applications

Employed in whole genome sequencing, resequencing, targeted sequencing, and whole exome sequencing.
Whole exome sequencing focuses on the coding regions (exons) representing about 2-3% of the genome.

Metagenomics & 16S rRNA Gene

16S rRNA is vital in prokaryotic ribosomes and widely conserved among bacteria.
Contains variable regions useful for evolutionary studies and species identification, acting as a diagnostic tool.

Detection of Nucleic Acids

Detection relies on base pairing through hybridization following high-temperature denaturation of DNA.
Southern blotting utilizes labeled DNA probes to detect complementary sequences in mixtures, while Northern blotting targets RNA.

Carbapenemases and Antibiotic Resistance

Carbapenemases such as OXA, KPC, and metallo-β-lactamases inactivate carbapenems by hydrolyzing the β-lactam ring.
β-lactams target bacterial cell wall synthesis by binding to transpeptidases, disrupting cell wall integrity leading to bacterial autolysis.

Gram-Negative Bacteria Structure

Distinctive outer membrane that includes lipopolysaccharide (LPS) as its outermost layer and a thin peptidoglycan layer on top of the plasma membrane.
Periplasmic space is present between the outer membrane and the inner plasma membrane.

Lipopolysaccharide (LPS) Composition

LPS consists of three parts: Lipid A (toxic), core polysaccharide (contains 9-12 sugars including KDO), and O antigen (long polysaccharide chain used for bacterial serotyping).
Lipid A anchors LPS to the membrane and is responsible for endotoxic activity, triggering immune responses and cytokine release.

Immunological Response to LPS

LPS is a potent immune stimulator that activates macrophages and dendritic cells, causing the release of pro-inflammatory cytokines (IL-1, IL-6, TNF).
While it triggers strong immune responses, it induces a poor antibody response making vaccine development challenging.

Porins and Outer Membrane Properties

Porins are protein complexes that allow passive diffusion of small, charged molecules through the outer membrane of Gram-negative bacteria.
Bacterial cell wall construction is reinforced by D-glucosamine, L-lysine, and D-alanine cross-links.

β-Lactam Resistance Mechanisms

Resistance to penicillin occurs through altered penicillin-binding proteins (PBPs) and reduced penetration due to fewer porins in Gram-negative bacteria.
β-lactamase enzymes, produced mainly by Gram-negative bacteria and some Gram-positive, confer resistance by hydrolyzing β-lactam antibiotics.

Types of β-Lactamases

Penicillinases target and inactivate penicillins.
Extended-spectrum β-lactamases (ESBLs) inactivate a broader range of β-lactams but typically not carbapenems.
Certain bacterial strains possess AmpC β-lactamases, often plasmid-encoded in "SPACE" organisms.

Characteristics of Gram-Positive Bacteria

Thick peptidoglycan layer retains the crystal violet stain used in Gram staining, distinguishing Gram-positive bacteria from Gram-negative.
Lipoteichoic acid is present and contributes to the structure of Gram-positive cell walls.

Bacterial Cell Structure and Function

Bacteria are unicellular prokaryotes lacking membrane-bound organelles and nuclei, with free nuclear materials.
Most possess unique cell walls containing peptidoglycan, a key target for both antibiotics and the immune system.

Surface Appendages and Bacterial Movement

Flagella, mainly composed of flagellin, facilitate movement towards nutrients in a process termed chemotaxis.
H antigen designation helps identify bacterial strains based on flagellar composition.

Capsules and Their Role

Capsules, composed mainly of polysaccharides, confer resistance to phagocytosis and contribute to virulence.
Weakly antigenic, capsules can induce IgM but fail to trigger class switching in antibody production.

Spore Formation in Gram-Positive Bacteria

Some Gram-positive bacteria enter a dormant spore state under nutrient scarcity, exhibiting resistance to heat and chemicals.
Dipicolinic acid within spores protects DNA; extremely resilient and can survive long periods of dormancy until favorable conditions return.

Bacteria Overview

Unicellular prokaryotes lacking nuclei and membrane-bound organelles.
Nuclear material is cytoplasmic; do not have mitochondria but contain an electron transport chain in membranes.
Most bacteria possess unique cell walls made of peptidoglycan, a key target for the immune system and antibiotics.

Bacterial Structure

Thick cell walls composed of peptidoglycan are crucial for structural integrity.
Gram staining differentiates bacteria: Gram-positive appears blue-purple; Gram-negative appears pink-red.

Gram Stain Process

Crystal violet is used to stain, followed by Gram's iodine as a mordant.
Alcohol treatment dehydrates the cell wall, trapping crystal violet in Gram-positive cells.
Counterstaining with safranin results in distinct coloration for Gram-positive and Gram-negative bacteria.

Gram-Positive Cell Wall

Features a thick peptidoglycan layer with lipoteichoic acid.
Retains Gram stain due to the dense structure.

Peptidoglycan Composition

Complex macromolecule consisting of glycan chains linked by peptide cross-links.
β-Lactam antibiotics mimic D-Ala-D-Ala in structure, inhibiting wall synthesis and leading to bacterial autolysis.

Antibiotic Resistance Mechanisms

Major mechanism in Gram-positive bacteria involves altered penicillin-binding proteins due to mutations.
Gram-negative bacteria resist through reduced penetration and β-lactamase enzymes that hydrolyze the antibiotic.

β-Lactamase Enzymes

Enzymes conferring resistance by inactivating β-lactam antibiotics; prevalent in both Gram-positive and negative bacteria.
Types include Penicillinases, Extended-Spectrum β-lactamases (ESBLs), and Carbapenemases.

Gram-Negative Cell Envelope

Comprises a unique outer membrane containing lipopolysaccharides (LPS), thin peptidoglycan, and periplasmic space.
LPS triggers strong immune responses, activating macrophages to release pro-inflammatory cytokines.

LPS Components

Lipid A: Endotoxic, anchors LPS in the outer membrane and elicits immune responses.
Core polysaccharide: Essential for LPS integrity and viability.
O antigen: Provides serotype specificity and is used in identification (e.g., EHEC O157:H7).

Biological Properties of LPS

Induces fever and shock; however, induces a weak antibody response.
LPS activates complement and coagulation cascades in severe infections.

Gram-Negative Peptidoglycan

Composed of 1-2 layers and cross-linked through direct peptide bonding, lacking pentaglycine bridges.
Thinner and more porous than Gram-positive peptidoglycan.

Plasma Membrane of Bacteria

Similar to eukaryotic membranes, containing transport proteins and respiratory enzymes.
Sites for peptidoglycan synthesis and outer membrane component production.

Surface Appendages

Pili (Fimbriae): Allow adherence to host cells; involved in pathogenicity and genetic exchange.
Flagella: Required for motility and chemotaxis, with H antigens useful for strain identification.

Capsule (Glycocalyx)

Thick, viscous layer providing resistance to phagocytosis; poorly antigenic.
Composed of polysaccharides or proteins; examples include Streptococcus pneumoniae and Bacillus anthracis.

Bacterial Spores

Dormant state enabling survival under harsh conditions; resistant to heat and chemicals.
Germinate into growing cells when conditions improve; require autoclaving for sterilization.
Notable spore-forming bacteria include Bacillus and Clostridium species.

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Description

This quiz explores the mechanisms of RNA interference (RNAi), focusing on post-transcriptional gene silencing through miRNAs and siRNAs. It delves into the roles these small RNA molecules play in regulating protein expression and their implications in diseases like cancer. Test your understanding of RNA-based gene regulation and its biological significance.