Review for Exam 4 - Comprehensive Notes on Transposons and CRISPR

Exam 4 Review: Key Concepts and Details

General Exam Information

Exam 4 covers all material from the regulation of gene expression through the lecture given on Friday.
Questions will be drawn from every lecture, and the exam will not focus on one specific area.
Read each question carefully to ensure you understand what is being asked before answering.
Answer the question that is asked, not what you assume is being asked.

Transposons (Jumping Genes)

Transposons are also known as "jumping genes".
They are made of DNA and can move around in the genome.
They code for the transposon itself and the enzymes needed for their movement.

Class 2 Transposons (DNA Transposons)

Use a "cut and paste" mechanism.
Code for transposase, an endonuclease that cuts the transposon out.
The transposase recognizes the transposon, cuts it out, and inserts it elsewhere in the genome.
It also matches flanking direct repeats to ensure proper insertion.

Class 1 Transposons (Retrotransposons)

Use a "copy and paste" mechanism via an RNA intermediate.
Code for all necessary enzymes, including endonuclease and reverse transcriptase.
The enzymes are translated in the cytoplasm and then return to the nucleus.
The transposon is transcribed, reverse transcribed, and replicated into double-stranded DNA.
The new DNA copy is inserted elsewhere in the genome, while the original remains in place.

Barbara McClintock's Work with Corn

McClintock studied transposons in corn kernels, considering each kernel as an individual organism.
Multiple genes influence a single trait (color).
The system involves multiple genes acting on one trait: color.

Key Genes

C gene/ C prime (c/c'): Dominant C' leads to colorless phenotype; recessive cc is required for color production.
Bz gene (Bz/bz): Dominant Bz produces purple color; recessive bz produces brown color.
Ds (Dissociator): The transposon itself, coding for transposases and endonucleases.
Ac (Activator): Required for the Ds transposon to move.

Phenotype Determination

If the genotype is c’c’, the kernel will be colorless, regardless of the Bz genotype, because the paintbrush is absent.
A genotype of cc allows color to be expressed (purple with Bz allele, brown with bz allele).

Trihybrid Cross Example

A trihybrid cross (c’c’ Bz/Bz Ds/Ds x cc bz/bz ds/ds) was performed.
The male parent was colorless, potentially purple (but not visible), and had the Dissociator (Ds) element.
The female parent was capable of making brown pigment but lacked the Dissociator (ds/ds).
The resulting triploid organism in the F1 generation was heterozygous for all three genes (c’/c/c Bz/bz/bz Ds/ds/ds).
The C' allele masks the cc, resulting in a colorless phenotype.
The Bz allele masks the bz/bz, so purple color is produced; the color is not visible unless the transposon moves.
If Ac is activated, Ds can move, disrupting the C’ gene and allowing color to be expressed.
The timing of Ds movement affects the color pattern.

Color Patterns

Early movement leads to solid purple color.
Later movement leads to spotted patterns.
The later the movement, the fewer spots of color.

Key Takeaways on Transposons

Transposons are DNA sequences that move within the genome.
They code for the enzymes needed for their movement.
Class 2 use a cut and paste mechanism, Class 1 use a copy and paste mechanism.
Color expression indicates the activator is on – no movement can occur without it.

Reverse Transcriptase

Reverse transcriptase reverses transcription (RNA to DNA).
Transposons must undergo transcription to produce the enzymes (reverse transcriptase, nuclease, etc.) needed for movement.
These enzymes are translated in the cytoplasm and then transported back into the nucleus via nuclear localization signals.
In retrotransposons, the original transposon remains, and a copy is inserted elsewhere.
Transcription creates RNA, which is then reverse transcribed back into DNA by reverse transcriptase.
DNA polymerase replicates the single-stranded DNA to form double-stranded DNA.
Other enzymes like endonucleases and helicases aid in the insertion process.

Balanced vs. Unbalanced Transposition

Class 2 transposons are balanced because they involve cutting and pasting, so no DNA is gained or lost.
Class 1 retrotransposons are unbalanced because the original remains and a copy is also made.

Autonomous vs. Non-Autonomous Transposons

Autonomous: Can function independently.
Non-autonomous: Require assistance from other elements.
In mammals, the class 1 are nonlong terminal repeats because they lack repeat domains.

Autonomous – LINES (Long Interspersed Nuclear Elements)

Longer because they contain the gene for reverse transcriptase.
Make their own reverse transcriptase.

Non-Autonomous – SINES (Short Interspersed Nuclear Elements)

Require reverse transcriptase from LINES to move.
Utilize the same copy and paste mechanism as autonomous transposons.
SINE RNA contains specific sequences that reverse transcriptase recognizes, including terminal inverted repeats.

Transposon Function

Transposons code for the enzymes needed for their movement and for the transposon DNA itself.
They do not code for proteins other than those enzymes.
The enzyme DNA moves with the transposon to maintain functionality.

Allele Element (ae1)

A type of transposon that has DNA sequences at each end that can be cut by the restriction enzyme AluI (though mammals don't have these).
It codes for transposase.
It serves as a restriction site for AluI.
The sequence is AGCT (5’ to 3’ on one strand, with the complementary sequence on the other strand).

Impact on Gene Expression

Can insert into a gene, generating a new splice site.
Insertion can turn a normal sequence into an intron.
Results in a new exon.
Potentially creates new protein products, increasing genetic diversity.

Transposons and Gene Expression Control

Transposons may be a mechanism for controlling gene expression.
The exact mechanisms are not fully understood.

CRISPR Plasmid Components

CRISPR-Cas9 gene
Guide RNA gene and design (must be specific to the target DNA).
Promoter regions to drive transcription of the CRISPR-Cas9 gene and guide RNA.
Nuclear localization signal to ensure the plasmid enters the nucleus.
Optional: Reporter genes can also be added, such as GFP or antibiotic resistance genes.

Essential Components

Guide RNA (correctly designed).
Cas9 gene.

Wine Example

Illustrates how transposons cause color variation in grapes.

Anthocyanin

The gene codes for a color protein called anthocyanin.

Original Grapes

Had a functional anthocyanin gene for deep red color.

Deep Red Grapes

Deep, dark, rich red due to functional anthocyanin protein.
Used to make heavier Cabernet wines.

Transposon Insertion (Egress one)

Transposon inserted in front of the anthocyanin gene, disrupting the promoter region.
Turned off the gene, resulting in colorless grapes used for Chardonnay wines.

Deletion Event

A deletion occurred, taking out much of the transposon but leaving behind about 20-25 base pairs directly in front of the anthocyanin gene.
Slightly disrupted transcription of the anthocyanin gene, resulting in lighter red grapes.

Resulting Grapes

Created lighter red grapes, used to make rose wines.

MicroRNA (miRNA)

Genes code for microRNA (miRNA).

Process

miRNA gene is transcribed in the nucleus.
The miRNA forms a hairpin structure due to complementary base pairing.
The hairpin structure is transported to the cytoplasm.
One end of the hairpin is cleaved off.
Proteins bind to one strand of the miRNA, protecting it from degradation.
The unprotected strand is degraded by nucleases, resulting in single-stranded miRNA.

Mechanism

Single-stranded miRNA binds to messenger RNA (mRNA) if there is sufficient base pairing.
This creates a small region of double-stranded RNA, which is not recognized by ribosomes.
Translation is blocked because the ribosome cannot read the double-stranded region leading to a shorter polypeptide chain.