Podcast
Questions and Answers
What is the main function of Cre in the context of loxP sites?
What is the main function of Cre in the context of loxP sites?
Transposases only function through a 'cut-and-paste' mechanism.
Transposases only function through a 'cut-and-paste' mechanism.
False
What is the role of transposon mutagenesis in genetic studies?
What is the role of transposon mutagenesis in genetic studies?
To identify essential genes by capturing insertion sites.
The insertion of a transposon into DNA is facilitated by a ______________ mechanism.
The insertion of a transposon into DNA is facilitated by a ______________ mechanism.
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Match the following terms with their descriptions:
Match the following terms with their descriptions:
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What is the primary mechanism involved in the movement of LTR retrotransposons?
What is the primary mechanism involved in the movement of LTR retrotransposons?
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Non-LTR retrotransposons, such as LINEs, are considered non-autonomous elements.
Non-LTR retrotransposons, such as LINEs, are considered non-autonomous elements.
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What catalyzes the recombination during co-integrate resolution?
What catalyzes the recombination during co-integrate resolution?
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What is the main function of transposase in DNA-only transposons?
What is the main function of transposase in DNA-only transposons?
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The synthesis of long terminal repeat (LTR) element DNA from element RNAs involves converting retroviral RNA to _____ for preserving the genome.
The synthesis of long terminal repeat (LTR) element DNA from element RNAs involves converting retroviral RNA to _____ for preserving the genome.
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Non-autonomous elements require their own transposase for movement.
Non-autonomous elements require their own transposase for movement.
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Match the following transposable elements with their characteristics:
Match the following transposable elements with their characteristics:
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Which element requires an intermediate RNA for transposition?
Which element requires an intermediate RNA for transposition?
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What does LTR in transposons stand for?
What does LTR in transposons stand for?
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Integrase plays a critical role in joining DNA-only elements by cut and paste insertion.
Integrase plays a critical role in joining DNA-only elements by cut and paste insertion.
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SINEs depend on _____ proteins for transposition.
SINEs depend on _____ proteins for transposition.
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What are the crucial residues found in the catalytic cores of DNA-only transposases and retroviral integrases that are essential for transposition?
What are the crucial residues found in the catalytic cores of DNA-only transposases and retroviral integrases that are essential for transposition?
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Match the type of transposable element with its characteristics:
Match the type of transposable element with its characteristics:
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Which of the following is a characteristic of transposition proteins encoded by LTR elements?
Which of the following is a characteristic of transposition proteins encoded by LTR elements?
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Retroviral-like elements are capable of extracellular transmission.
Retroviral-like elements are capable of extracellular transmission.
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What occurs during the excision phase of DNA-only cut and paste transposition?
What occurs during the excision phase of DNA-only cut and paste transposition?
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Study Notes
DNA Mobility
- DNA mobility refers to the movement of DNA elements within a genome
- This movement impacts genome structure, function, and evolution
- Some mobile elements use RNA as an intermediate, while others use only DNA
- Mobility can be random or directed
- Mobile elements have been exploited for experimental mutagenesis and gene transfer
Mobile Genetic Elements
- Mobile genetic elements (MGEs) are common in all life forms (prokaryotes, archaea, and eukaryotes)
- They drive genomic plasticity
- MGEs influence genome structure, function, and evolution
- Some MGEs move through RNA intermediates; others only use DNA
- Their movement can be random or programmed
- Scientists use them in experimental mutagenesis and gene transfer
Transposition
- Transposition is the movement of a discrete DNA element to different target sites within a genome
- Two common mechanisms:
- excision and integration
- replication
- The movement is facilitated by proteins that are specific to the particular element
- Transposition often involves the duplication of the target DNA sequence, a "target site duplication" (TSD).
Transposable Elements and Genetic Diversity
- Transposable elements (TEs) are major sources of genetic diversity
- TEs disrupt genes within and between chromosomes, potentially altering gene expression
- TEs can alter gene expression by introducing regulatory signals near host genes (promoters, enhancers, splice sites, polyA sites)
- TEs are substrates for homologous recombination, influencing genome dynamics
- Transposable elements are highly dynamic parts of genomes
Transposons and Mutations
- Transposons are a type of transposable element that causes mutations
- Barbara McClintock studied transposons in maize, demonstrating how their movement affects pigment expression
Active Human Transposons
- Researchers identified active human transposons from mutations in patients
- Haig Kazazian identified insertions in the Factor VIII gene in unrelated boys
- The insertions were not in their parents, indicating recent activity of transposable elements
- The inserted sequences were non-LTR LINE elements
Frequency of Transposon Insertion
- Transposon insertion frequency varies across species:
- 0.3% in humans, where TEs comprise about 45% of the genome
- 10% in mice, where TEs comprise about 38% of the genome
-
50% in Drosophila, where TEs comprise about 5.5% of the genome.
- Most insertions in humans and mice are non-coding, relatively inconsequential
Classification of Transposable Elements
- Transposable elements (TEs) are categorized based on various criteria, including structure, transposition mechanism, and effects on the donor site.
- Major groups are defined by structure (DNA-only, RNA intermediates, LTR, non-LTR elements)
Genome Components in Different Organisms
- The slides present a comparative chart reflecting the relative abundance of various transposable elements in bacterial, archaeal, and eukaryotic genomes, in different species (e.g. humans, mice, yeast, maize, Drosophila).
DNA-only Transposons
- DNA-only transposons move via cut-and-paste or replicative mechanisms
- Terminal inverted repeats (TIRs) are essential for their movement
Long Terminal Repeat (LTR) Elements
- These elements exhibit an RNA-DNA intermediate cycle
- LTRs facilitate RNA to cDNA conversion for integration into the host genome
- Retroviruses disseminate extracellularly, using an envelope protein
- Retroviral-like elements replicate intracellularly
Non-LTR Elements
- LINEs (Long Interspersed Elements) are autonomous, possessing their transposing machinery
- SINEs (Short Interspersed Elements) lack their own transposition machinery and rely on LINEs
- Both LINE and SINE elements occupy a significant portion of the human genome
DNA Transposon Superfamilies
- A variety of distinct transposon superfamilies are highlighted, including bacterial (IS elements, Mu, Tn elements) and eukaryotic families (Mutator, Tc1/mariner, hAT, piggyBac).
DNA-only Cut and Paste Elements
- IS elements contain inverted repeats flanking a transposase gene
- Composite transposons comprise multiple IS elements flanking other genes
DNA-only Cut and Paste Transposition Strategy
- The strategy involves transposase binding, DNA cleavage, excision, target joining, gap repair, leading to TSD formations
Excision Pathways and 3' OH generation
- Various pathways by which transposases carry out excision, producing a 3'OH end that's critical for subsequent reactions
DNA breakage by Transposase
- Transposase-mediated DNA breakage occurs in trans, requiring the specific binding and catalytic domains of the transposase.
RAG and VDJ Recombination
- RAG and VDJ recombination systems employ nicking and attack mechanisms to liberate spacers from the intermediate segments generating 3'OH DNA ends, similar to transposon activity.
Replicative Nick and Paste Transposition
- This process involves the replication of the mobile element before inserting a copy at a new site.
Co-integrate Resolution
- Co-integrate resolution involves using conservative site-specific recombination, a specific recombination method using enzymes called resolvases.
LTR Elements
- Retrotransposons (Ty and Gypsy) and retroviruses (MuLV, RSV, HIV) use an RNA intermediate in their transposition cycle.
LTR Retrotransposons – Intracellular Propagation
- The processes involve assembly, translation, reverse transcription, and integration (using integrase) to ensure the propagation of LTR retrotransposons.
Retroviruses – Intercellular Propagation
- Retroviruses use RNA as an intermediate, with processes involving integration and packaging into new viral particles to propagate intercellularly
LTR Elements (cont)
- The RNA intermediate produced during retrotransposition contains information from both 5' and 3' LTRs
Synthesis of Long Terminal Repeat (LTR) Element DNA
- The synthesis of LTR DNA from RNA depends on reverse transcription. This process preserves the integrity of the retroviral genome.
Integration of CDNA Intermediates
- Integrase facilitates the integration of cDNA intermediates into the host genome.
Structural Similarity
- HIV-1 integrase and MuA transposase exhibit structural similarities, particularly in their catalytic cores. Key residues in integrase and transposase catalyze DNA cleavage, which is critical.
Non-LTR Retrotransposons
- LINEs (Long Interspersed Elements) and SINEs (Short Interspersed Elements) are two primary classes of Non-LTR retrotransposons. LINEs are autonomous and SINEs are non-autonomous.
Transposition of Non-LTR elements
- Non-LTR element transposition generally involves RNA intermediates and reverse transcription.
Non-LTR Retrotransposons (cont.)
- Group II introns are self-splicing mobile genetic elements (MGEs).
Conservative Site-Specific Recombination (CSSR)
- This process involves site-specific recombinases without affecting DNA sequence length and relying solely on short DNA sequences and recombinases. Recombinases carry out recombination at specific sites
Recombination
- Recombination mechanisms are illustrated and classified based on the homology (direct or inverted repeats) required between DNA sequences.
The λ Integrase family
- Tyr recombinases of site-specific recombinases, more than 100 such enzymes are known. Examples: bacteriophage λ, P1 phage Cre, and S. cerevisiae Flp systems.
Resolution of P1 Replicative Intermediates
- This process involves resolvase and loxP sites that yield two separate DNA molecules.
A 34-bp loxP site
- This specific site sequence is essential for recombination with Cre recombinase.
Synapsed Substrates for Cre-mediated Recombination
- Graphical representations show Cre-mediated recombination substrates undergoing synapsis. These are the specific substrates forming the structure for Cre-mediated recombination.
Model for Cre-mediated Recombination
- Model illustrating Cre recombination, using Holliday junctions as the intermediate (structural components that facilitate recombination).
Transposase-mediated DNA Fragmentation
- Describes the process of transposase-mediated insertion and DNA fragmentation and the related methodology
ATAC-seq Chromatin Accessibility
- The technique involves Tn5 transposase mediated oligonucleotide insertions for genome-wide mapping of chromatin accessibility
- Graphical representations and related quantitative data are provided.
Nucleosome Mapping
- Shows how analysis of genomic fragments produced by a DNA-fragmenting transposase (from Tn5) helps identify the centers of nucleosomes in the cell.
Transposon Mutagenesis
- Transposon mutagenesis approaches for identifying and characterizing essential genes in various organisms, including mice and Candida glabrata
Cre-activated Transposon Mutagenesis
- An approach for discovering genes involved in cancer, using Cre-mediated transposon insertion for targeted mutagenesis and analysis.
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Description
This quiz explores key concepts in genetics, focusing on the functions of Cre proteins in relation to loxP sites and the role of transposon mutagenesis in genetic studies. Test your understanding of various genetic mechanisms and their applications.
