Exam 3 Genetics

Are you a transgenic organism? Based on the book's definition, why or why not? Based on the definition from class, why or why not?

Based on the books definition, yes, I am a transgenic organism. A transgenic organism is an organism that contains a gene from someone in the same or different species. Since I have genes from my parents it is evident that I am a transgenic organism.

What are the benefits of a binary UAS-Gal4 system?

It is very specific. Gal4 acts like a switch to turn on the gene of interest that is connected to UAS. When you cross the two flies, the UAS-linked gene is only turned on in the places where Gal4 is active.

Are you concerned about GMO alleles "leaking" out into non-GMO crops or wild plants? Do you think that GMOs are safe?

Yes, I think it is fair to be concerned about GMO alleles leaking out into non-GMo crops or wild plants. Through pollen, plants are able to reproduce with GMO plants even if it was never intended. As for safety, I think that all GMOs are tested and safe for use and eating. I think its important though to continue to research the cost of using and consuming GMOs long term.

What is the role of poly-linkers?

Fragment of DNA that contains restriction enzyme sites. Its role is to make it easier to insert a gene into the plasmid. It cuts out a specific sequence of the polylinker as well as a specific sequence of the gene and can combine them if their base pairs align. It provides flexibility for where the gene will end up.

In our generic vector, we have a selectable marker and the inserted gene. If the inserted gene is GFP, do we need to have miniwhite? What features of the promoter/enchancer sequence would cause you to say YES or NO?

- No, we don't need miniwhite if the GFP is strongly expressed and in an area that is easy to see. Ie: eyes, whole body, etc.

- Yes, we do need miniwhite if GFP is expressed at a specific time or in a specific neuron (tissue specific) that may be hard to see and track.

Vocab:

Transgene

is a gene that has been inserted into an organism, and it comes from another individual, either from the same species or a different species.

Plasmid

small circular piece of DNA in bacteria separate from chromosomes. Used as a tool to carry and insert foreign genes into an organism.

Polylinker

section of DNA in the plasmid that has restriction enzyme sites. Helps scientists easily insert a gene into a plasmid and then into the organism.

P-element

known as a jumping gene. Helps carry genes of interest into the genome by inserting itself into the fly genome (transposable element)

Transformation

is the process of introducing foreign DNA into a cell. Inserting new gene for the possibility of passing it to an offspring

NHEJ repairs double strand breaks that occur naturally in cells. 95% of the time, NHEJ repairs the double strand break (DSB) perfectly. NHEJ results in small indel mutations 5% of the time during the repair of normal DSBs. Researchers observe that CRISPR-Cas9 will produce indices 100% of the time when NHEJ is used to repair DSBs made by CRISPR-Cas9. Why?

Although NHEJ usually repairs natural double-strand breaks with high accuracy, it produces indels almost 100% of the time when repairing cuts made by CRISPR-Cas9. This is because Cas9 cuts at the exact same spot every time, and if the repair is perfect, Cas9 will cut again. Repeated cutting and repair eventually lead to insertions or deletions that prevent further cutting, making indel formation nearly guaranteed.

How many places in the fly genome will a 5-base cutter restriction enzyme cut? What about a 6-base cutter? A 7-base cutter?

Fly genome has 165 million base pairs

4^5= 1024 → (161,132)

4^6= 4096 → (40,283)

4^7= 16384 → (10,070)

What are the key differences between the bacterial CRISPR system and the engineered system used for eukaryotes?

In eukaryotes, CRISPR is engineered for gene editing, whereas in bacteria it functions as an immune defense. A key difference is that in eukaryotes, the Cas9 protein and guide RNA must be delivered into the cell's nucleus. Also, the engineered system uses a simplified sgRNA that doesn't require the processing steps seen in bacteria.

Is the PAM sequence found in the spacer or the repeat or not at all at a CRISPR locus?

The PAM sequence is located next to the target DNA in the foreign genome, but it is not part of the spacer or stored in the CRISPR locus. During spacer acquisition, the CRISPR system recognizes and cuts near the PAM, but only the adjacent foreign DNA sequence (not the PAM itself) is integrated into the CRISPR array.

What are the key features of the transgenic locus that makes CRISPR based mutagenic chain reaction work different?

The key feature of the transgenic locus in CRISPR-based mutagenic chain reaction (MCR) is that it can copy itself. Unlike normal CRISPR, which changes just one copy of a gene, MCR includes both the guide RNA and Cas9 in the DNA. When it cuts the other copy of the gene, the cell uses the MCR part to repair it, copying the whole system over. This makes the change spread quickly by turning heterozygotes into homozygotes.

how is mutagenic chain reaction different from normal CRISPR?

This is different from normal CRISPR because, in regular CRISPR, the tool edits only one copy of a gene and doesn't stay in the genome—it makes a change and is done. In contrast, CRISPR-based mutagenic chain reaction (MCR) builds the CRISPR system (Cas9 and guide RNA) into the DNA. This means it keeps cutting and copying itself into the other copy of the gene, spreading the change to all copies and passing it on more easily to future generations.

homology arms

DNA sequences on either side of a desired gene insert that match the target site, used to guide HDR.

CRISPR

gene editing tool that uses guide RNA to target specific sequences in DNA

Cas9

enzyme that cuts DNA at a specific site specified by the gRNA

PAM

the cut site right next to the target site that Cas9 needs to recognize to cut the DNA

NHEJ

non-homologous end joining- quick and error prone DNA repair method that joins cut ends without a template

HDR

homologous directed repair; A precise DNA repair process that uses a matching DNA template to fix breaks.

How does Sanger sequencing differ from Sequencing-By-Synthesis (Illumina)? How is the use

of terminator-nucleotides different between these methods?

Sanger sequencing and Illumina both use fluorescence, but they differ in how they stop DNA synthesis. In Sanger sequencing, ddNTPs permanently stop DNA synthesis, creating fragments that are later pieced together to determine the sequence. In contrast, Illumina uses reversible terminator nucleotides that temporarily block DNA polymerase. After each base is read, the block is removed, allowing sequencing to continue in a step-by-step, parallel fashion.

How is a sequencing vector different from a transgenic vector?

A transgenic vector is used when scientists want to insert a gene into an organism to give it a new trait, like making a plant resistant to pests. These vectors usually have extra parts, like promoters, to make sure the gene is turned on and works properly inside the organism. In contrast, a sequencing vector is much simpler and is only used in the lab to hold a small piece of DNA so its sequence can be read. It doesn't need to function in a living organism—just provide what's needed for sequencing, like primer binding sites.

If you were using molecular cloning to isolate a fragment of DNA for Sanger sequencing, how could you double check that your fragment successfully ligated into your vector (plasmid)?

You would transform bacteria with the ligation product and plate them on ampicillin-containing media; only those with the plasmid (with or without insert) will grow.

Restriction Enzyme

enzyme that cuts DNA at specific sequences

molecular cloning

A method used to copy and insert DNA fragments into plasmids for study or use.

PCR

A technique to quickly make many copies of a specific DNA segment.

happens in 3 steps

1. denaturation

2. annealing

3. amplification

sticky ends

Single-stranded DNA overhangs created by restriction enzymes that help DNA fragments stick together.

bridge amplification

A process used in Illumina sequencing where DNA strands bend and attach to nearby primers to form clusters of identical copies.

Why is a partial restriction digest necessary to make overlapping BAC clones?

A partial restriction digest is necessary to make overlapping BAC clones because it cuts the DNA at some but not all restriction sites. This creates large, overlapping fragments instead of cutting everything into small, non-overlapping pieces, which helps scientists assemble the full genome by aligning the overlapping regions. (incomplete digestion)

What features of a genome prevents genome assembly?

Genome assembly is made difficult by repetitive sequences, which make it hard to tell where reads belong, which can lead to uneven coverage

What is the principal difference between paired-end BAC libraries and selective BAC library

Strategies?

The principal difference is that paired-end BAC libraries are designed to give information about the distance and orientation between two ends of large DNA fragments, helping with genome scaffolding and assembly, while selective BAC libraries are focused on specific regions of the genome (like gene-rich areas or regions of interest) and are used to study those parts more closely.

What are two reasons you should make cDNA libraries from multiple tissues, ages, sexes

when performing gene annotation?

To accurately annotate a genome, it's important to make cDNA libraries from multiple tissues, ages, and sexes because gene expression varies across biological conditions. First, many genes are tissue-specific or expressed at different levels depending on the tissue, developmental stage, or sex, so sampling broadly ensures more complete gene coverage. Second, genes can undergo alternative splicing, meaning the same gene may produce different mRNA and proteins in different tissues. By sequencing cDNA from diverse sources, we can capture a wider variety of gene transcripts and better understand their expression and regulation.

Why can gene duplication lead to gene decay between gene copies? Why might gene

duplication leads to divergence between gene copies?

Gene duplication can lead to gene decay because once a second copy exists, one copy might no longer be essential and can accumulate mutations without harming the organism, eventually becoming a nonfunctional pseudogene. On the other hand, duplication can also lead to divergence, where one copy keeps the original function while the other develops new or specialized roles, allowing the organism to adapt or gain new abilities.

What are three ways you can infer the function of genes?

Gene function can be inferred by sequence similarity to known genes, expression patterns across tissues or conditions (e.g., using cDNA libraries), and evolutionary conservation—genes that are conserved across species are likely to have important functions. Additionally, studying gene families or the effects of gene knockouts can also provide strong functional clues.

Are members of a gene family within a species (e.g., hemoglobin) homologous?

Yes, members of a gene family within a species are homologous because they come from a common ancestral gene.

Shotgun sequencing

A method that breaks the genome into random small fragments, sequences them, and then assembles the pieces back together.

BAC library

A collection of bacterial cells, each carrying a large piece of DNA, used to map and sequence genomes.

Homolog

A gene related to another by shared ancestry, either within the same species or across different ones.

Alignment

The process of matching up DNA, RNA, or protein sequences to identify regions of similarity.

ORF (Open Reading Frame)

A stretch of DNA with no stop codons that could potentially code for a protein.

cDNA

A DNA copy made from mRNA that represents the coding regions (exons) of expressed genes.

Protein domain

A distinct part of a protein that has a specific structure or function and can evolve independently.

Motif

A short, conserved sequence pattern in DNA or protein that is often linked to a specific function.

Gene family

A group of similar genes that evolved from a common ancestor and often perform related functions.

Divergence

The process by which duplicated genes or species accumulate differences over time.

Degradation

The breakdown or loss of function in a gene, often leading to the formation of a pseudogene.

1. Insertion

The addition of one or more base pairs into a DNA sequence. Can disrupt genes or regulatory regions.

2. Deletion

The loss of a segment of DNA. Often more harmful than insertions; may remove essential genes.

3. Inversion

A segment of a chromosome is flipped and reinserted in reverse order. Can suppress recombination and lead to the formation of supergenes.

4. Translocation

A segment of one chromosome is moved to a different chromosome.May disrupt gene function or regulation if it breaks genes or alters their context.

5. Aberrant Recombination

Improper or abnormal crossing over during meiosis. Can cause duplications, deletions, inversions, or translocations.

6. Read Depth

The number of times a DNA region is sequenced in next-generation sequencing (NGS). Used to detect copy number variations — high read depth = duplication; low = deletion.

7. Supergenes

A group of neighboring genes inherited together due to suppressed recombination (often caused by an inversion).

1. Illumina sequencing (NGS) can identify large insertions or deletions by assessing read depth. How does read depth relate to copy number?

Read depth reflects copy number because duplicated regions produce more reads, while deleted regions produce fewer.

2. How do inversions prevent recombination?

Inversions prevent recombination by forming inversion loops during meiosis, which lead to abnormal crossover products and nonviable gametes.

3. How can you detect duplications with PCR?

Duplications can be detected with PCR by designing primers around the suspected duplicated region. If a duplication is present, the PCR product will be longer than expected. Running the PCR products on a gel will show this difference in size — duplicated regions produce larger bands.

4. How do inversions generate supergenes?

Inversions suppress recombination within a region, which keeps groups of genes inherited together. Over time, these tightly linked genes can evolve to function as a unit

5. Which do you think are more likely to be harmful to human health, duplications or deletions?

Deletions are more likely to be harmful because they can remove important genes that are essential for survival or normal function and can cause haploinsufficiency. Losing a gene usually has a more severe effect than having an extra copy.

1. What is the principal difference between cut-and-paste vs. copy-paste TEs?

Cut-and-paste TEs (DNA transposons): Move by physically excising from one spot and inserting into another. Copy-paste TEs (retrotransposons): Copy themselves via an RNA intermediate and insert the copy elsewhere, leaving the original behind.

2. Are transformation-plasmids used in genetic engineering autonomous or non-autonomous?

Non-autonomous. They require a source of transposase (often provided separately) because they can't move on their own.

3. Would P-element transformation work better in a P-background or an M-background or both?

M-background. M-type flies don't produce repressive piRNAs, so P-elements can be activated. In P-type flies, piRNAs suppress P-element movement.

4. Do genes or TEs make a larger contribution to variation in genome size across the tree of life?

TEs (Transposable Elements) contribute much more. Genome size variation is largely due to TE expansion, not differences in gene number.

Transposon

A DNA element that can move to new positions in the genome.

Retrotransposon

A TE that uses RNA and reverse transcription to insert copies elsewhere.

Transposase

An enzyme that catalyzes the movement of DNA transposons.

LTR (Long Terminal Repeat)

Repeated sequences at both ends of some retrotransposons.

Inverted Repeat

Short, mirror-image DNA sequences at both ends of a DNA transposon.

1.Does methylation always shut down (reduce) expression of a protein-coding gene?

Not always, but often. Methylation at promoter regions usually reduces gene expression, but effects can vary by context.

2. What is the female P-type fly doing to the egg to protect the offspring from TE attack?

She deposits piRNAs into the egg. These piRNAs recognize and silence P-elements, protecting the next generation.

3. What is a similarity between the piRNA system and CRISPR?

Both use small RNAs as guides to silence foreign genetic elements — piRNAs for TEs, CRISPR for viruses.

4. If you wanted to perform P-element insertion into Drosophila for transgenesis, should you use an M-cytotype or a P-cytotype?

M-cytotype. It lacks repressive piRNAs, allowing P-element activity.

5. Explain body size differences between Tigons and Ligers.

Ligers (lion dad × tiger mom) are huge because they inherit growth-promoting genes from the father and don't receive maternal regulation. Tigons (tiger dad × lion mom) are smaller — both parents contribute growth-limiting signals.

CpG

A cytosine followed by guanine in DNA; common site for methylation.

Methylation

Addition of a methyl group to DNA; often silences gene expression.

Silencing

The turning off of gene expression, often via methylation or piRNAs.

Epigenetic Inheritance

Passing on gene expression patterns without changing DNA sequence.

piRNA

Small RNAs that silence transposable elements in the germline.

1. Why haven't all genes from the mitochondria moved to the nuclear genome?

Not all mitochondrial genes have moved to the nuclear genome because some of them need to stay close to where the energy production happens—in the mitochondria. These remaining genes often make proteins that are part of the electron transport chain, which is super sensitive and needs to be controlled quickly and locally.

2. What are examples of insect cellular endosymbiosis that affect human health (you'll have todo your own research for that one).

Some insects have tiny bacteria living inside their cells—this is called cellular endosymbiosis. These bacteria can actually influence how the insects behave or function in ways that affect human health. Mosquitoes and Wolbachia: Some mosquitoes carry a bacteria called Wolbachia. This bacteria makes it harder for the mosquitoes to carry and spread viruses like dengue and Zika, which are harmful to humans. Scientists are even releasing Wolbachia-infected mosquitoes to help reduce disease transmission.

3. Why is a smaller genome beneficial to some mitochondria?

A smaller genome also makes replication faster and less energy-demanding, which is super important for mitochondria since their main job is to produce energy for the cell. Less DNA means fewer resources are needed to copy it, helping mitochondria stay efficient and responsive, especially in high-energy-demand cells like muscle or nerve cells.

4. What are the consequences of heteroplasmy?

can lead to unpredictable effects on health. Depending on the ratio of mutated to healthy mitochondria, it can cause mild to severe diseases, especially in energy-hungry organs like the brain, heart, or muscles.

5. Why does recombination (and thus sex) required to alleviate the effects of Muller's Ratchet?

Recombination, which happens during sex, helps mix and match genes from two parents. This allows harmful mutations to be separated from healthy genes, so offspring have a better chance of inheriting fewer bad mutations. Without recombination, like in asexual or non-recombining systems, harmful mutations build up over time because there's no way to "shuffle" them out

mtDNA (mitochondrial DNA)

The small, circular DNA found in mitochondria, inherited separately from the nuclear DNA.

Uniparental inheritance

When genes (like mtDNA) are inherited from only one parent—usually the mother in humans.

Heteroplasmy

A condition where a cell has a mix of normal and mutated mtDNA, which can affect how well mitochondria work.

Endosymbiosis

A relationship where one organism lives inside another; mitochondria are believed to have started as free-living bacteria that were engulfed by early cells.

Muller's Ratchet

A process where harmful mutations build up in DNA over time when there's no recombination to remove them.

Mitotic drift

The random changes in the proportion of mutated vs. normal mtDNA during cell division, which can lead to different effects in different tissues.