Molecular Techniques and Evolution

Question 1

Briefly describe the steps of recombinant DNA technology.

Answer 1

Vector + DNA fragment —> recombinant DNA –> replication of recombinant DNA within host cells —> isolation, sequencing, and manipulation of purified DNA fragment.

Question 2

__________ enzymes and DNA ______ allow insertion of DNA fragments into cloning vectors.

Answer 2

Restriction; ligases

Question 3

What is EcoRI?

Answer 3

A restriction enzyme that makes staggered cuts at the specific 6-bp palindromic sequence GAATTC, yielding fragments with single-stranded complementary 4-base “sticky” ends.

Question 4

What is ligase’s role in DNA recombination?

Answer 4

Ligase (links fragments during DNA replication) links DNA fragments with either sticky end or blunt ends into vector DNA.

Vector DNA sticky ends base-pair only with the complementary sticky ends on the genomic DNA EcoRI fragment.

Question 5

(T/F) T4 DNA ligase covalently joins the sugar-phosphate backbones on each strand.

Answer 5

True!

Question 6

What are plasmids?

Answer 6

Circular, double-stranded DNA molecules that replicate separately from a cell’s chromosomal DNA.

Question 7

What are the three things that plasmid vectors contain?

Answer 7

1) A synthetic polylinker region containing the only copy of several restriction enzyme recognition sequences, into which an exogenous DNA fragment can be ligated.

2) A selectable gene, such as AMPR, encoding the enzyme B-lactamase, which confers resistance to ampicillin.

3) A replication origin (ORI) sequence where DNA replication can be initiated by host-cell enzymes.

Question 8

Match the following terms to their definitions regarding DNA cloning in a plasmid vector.

1) Vector preparation
2) Transformation
3) Selection
4) Plasmid regulation

A) Some E.coli cells that are mixed with recombinant vector DNA and subjected to a stress such as heat shock will take up the plasmid DNA.

B) Plasmid DNA replicates and segregates into daughter cells, forming an ampicillin-resistant colony from proliferation of each cell containing the cloned DNA. All colony cells contain plasmids with the same inserted DNA.

C) A DNA fragment is ligated into the POLYLINKER region of a plasmid vector containing an ampr

D) Only the cells containing the plasmid and expressing B-lactamase survive on ampicillin-containing medium

Answer 8

Vector preparation: A DNA fragment is ligated into the POLYLINKER region of a plasmid vector containing an ampr

Transformation: Some E.coli cells that are mixed with recombinant vector DNA and subjected to a stress such as heat shock will take up the plasmid DNA.

Selection: Only the cells containing the plasmid and expressing B-lactamase survive on ampicillin-containing medium

Plasmid regulation: Plasmid DNA replicates and segregates into daughter cells, forming an ampicillin-resistant colony from proliferation of each cell containing the cloned DNA. All colony cells contain plasmids with the same inserted DNA.

Question 9

How can you screen for successfully ligated plasmids with gene of interest?

Answer 9

Screening involves alpha-complementation.

Bacterial cells are able to make both subunits of beta-galactosidase; from LacZ omega gene and LacZ alpha gene. Beta-galactosidase can digest X-gal and the bacterial cells become blue.

If the insert is ligated with the vector, LacZ alpha gene is disrupted, only lacZ omega gene is made and there is no alpha-complementation. The cells are white.

If the insert is not ligated with the vector, vector is going to be transformed into bacteria where both subunits are made. The cells are blue.

Question 10

What are the three major steps of PCR? What are its three key components?

Answer 10

Major steps
1) Denaturation: separating target DNA strands
2) Annealing of primers
3) Extension process of replication
*repeat cycle many times to amplify

Key components:
1) Taq DNA polymerase
2) dNTPs
3) DNA template

Question 11

(T/F) Like in vitro replication, we can also do in vitro transcription.

Answer 11

True!

Question 12

RNAi (RNA interference) with double-stranded RNA causes an __________ of specific gene function.

Answer 12

Inhibition

*this allows us to study the function of the gene

Question 13

How is siRNA produced in vitro?

Answer 13

Two plasmid vectors containing the target gene coding sequence in OPPOSITE orientations adjacent to a promoter.

Then, transcription of both vectors occurs using RNA POLYMERASE and ribonucleoside triphosphates. This yields many RNA copies in both orientations, which hybridize to form dsRNA.

This dsRNA is injected into cells and cleaved by DICER into siRNAs.

Question 14

Why does RNA interference (RNAi) produce siRNAs and not miRNAs in vitro?

Answer 14

The vectors have complementary gene sequence and they hybridize forming 100% complementary base pairs. miRNAs do not have 100% complementary base pairs, but siRNAs do.

Question 15

What is the mechanism for RNAi to inhibit gene function?

Answer 15

After producing the siRNAs, RISC mediates recognition and hybridization between one strand of the siRNA and its complementary target mRNA sequence.

Then, specific nucleases in the RISC cleave the mRNA-siRNA hybrid.

No gene to translate from; thus no gene function.

Question 16

What is shRNA? How/why is it made?

Answer 16

Double-stranded small hairpin RNA (shRNA)

Instead of inserting a huge part of gene of interest to make dsRNA, you can insert a hairpin construct, which contains a tandem arrangement of both sense and antisense sequences of the target gene.

This is transcribed to shRNA that is cleaved by Dicer to form siRNA.

It can be used for precise targeting.

Question 17

What does CRISPR stand for?

What does Cas stand for?

Answer 17

Clustered, Regulatory Interspaced Short Palindromic Repeats.

CRISPR associated proteins

Question 18

What does the CRISPR-Cas do in bacteria and archaea?

Answer 18

CRISPR-cas constitute an RNA-mediated defense system in bacteria and archaea which protect them against viruses and plasmids.

Question 19

Match the following steps of the CRISPR-mediated immunity done in bacteria and archaea:

1) Step 1
2) Step 2
3) Step 3

A) CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus, processed, and bind to the Cas protein.

B) The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade the nucleic acid.

C) A copy of the invading nucleic acid is integrated into the CRISPR locus.

Answer 19

Step 1: A copy of the invading nucleic acid is integrated into the CRISPR locus.

Step 2: CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus, processed, and bind to the Cas protein.

Step 3: The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid (even in the future) and the Cas proteins degrade the nucleic acid.

Question 20

Single nucleotide mutations can be introduced into the genome using an engineered ________________ system.

Answer 20

CRISPR-Cas9

Question 21

How can you engineer a CRISPR-Cas9 system?

Answer 21

Construct a plasmid encoding Cas9 and another plasmid encoding the guide RNA (guides Cas9 to the target of the gene of interest).

Express these components by transfection with plasmids or by direct injection of Cas9 mRNA and guide RNA.

Question 22

What are the two components of the guide mRNA?

Answer 22

1) A sequence that folds into a hairpin scaffold structure that binds Cas9.

2) A sequence of 20 nts corresponding to the targeted site in the gene.

Question 23

After finding the region of interest, what dose Cas9 do (aka its mechanism)?

Answer 23

Base pairing between guide RNA and its complementary genomic DNA sequence directs Cas9 complex to the targeted region of the genome.

Two distinct Cas9 nuclease activities CLEAVE both strands of the target DNA adjacent to the heteroduplex formed with the guide RNA.

Question 24

What are the two possibilities to repair-target gene inactivation (due to cleavage of both strands by Cas9)?

How do these introduce mutations (which kind)?

Answer 24

1) Nonhomologous end joining (NHEJ): repairs the ds target gene cleavage, but removes a few bases at the cleavage site, inactivating gene function due to FRAMESHIFT MUTATION.

2) Homology-directed repair (HDR): inclusion of a ~100 nt single-stranded DNA segment that spans the sequences flanking the cleavage site along with the Cas9 mRNA and the guide RNA causes homologous recombination repair, which can introduce SINGLE BASE CHANGES in the repaired genomic DNA.

Question 25

While RNAi _______ BCL11A, CRISPR _______ BCL11A to treat sickle cell disease and B-thalassemia.

Answer 25

Silences; Mutates

Question 26

Briefly describe how silencing/mutating BCL11A treats sickle cell disease and B-thalassemia.

Answer 26

Our hemoglobin is not the same as when we are fetus vs when we are born.

When we go from fetus to a baby, our α subunits are maintained, the γ subunits decrease and our β subunits increase. This is all done by BCL11A protein, which represses fetal subunits from being produced.

In these disorders, the β subunits are mutated but the fetal subunits are fine.

By blocking the repressor protein (BCL11A) by silencing or mutating the gene, we can reactivate the fetal subunits in the new born.

Question 27

What is the difference between homology and deep homology?

Answer 27

Homology is similarity due to shared ancestry between a pair of structures or genes in different taxa.

Deep homology: whereas homology is seen in the pattern of structures such as limb bones of mammals that are evidently related, deep homology can apply to groups of animals that have quite dissimilar anatomy.

For example, the human heart and the heart of a fly are analogous in function and look totally different, yet they are made in a very similar way with an ancient heart GRN (gene regulatory network).

Question 28

(T/F) The different pigmentation patterns of flies evolved due to the absence, presence, and identity of enhancers that changes where the pigmentation gene is expressed and when it is expressed during wing development.

Answer 28

True!

Evolution of enhancers can lead to the expression of genes in different places and different times.

Question 29

What are the three possibilities of duplicating genes with four different functions?

Answer 29

1) Degeneration/gene loss: one paralog retains the full 4 functions, the other has none.

2) Subfunctionalization: can generate paralogs that has partial functions (example 2 in each) of the ancestor, enabling them to function, be regulated, and evolve independently.

3) Neofunctionalization: can lead to the generation of one paralog with all the functions and a paralog with NEW biochemical functions.

Question 30

While natural selection, drift, and gene flow control flow of genomic variation, ____________ and _____________ are the source of genomic variation.

Answer 30

Mutation; Recombination

Question 31

What are the two methods to demonstrate common descent?

Answer 31

1) Paleontological (fossil record)
2) Molecular (sequence conservation)

Question 32

What does LUCA stand for?

Answer 32

Last universal common ancestor

Question 33

How many protein families trace to LUCA by phylogenetic criteria?

Answer 33

355 protein families

Question 34

Based on the 355 protein families that trace back to LUCA, what was the predicted environment?

Answer 34

Aerobic
CO2-fixing
H2-dependent with wood-ljungdalh pathway
N2-fixing
Thermophilic

Question 35

There is a hypothesis that before LUCA, there was an RNA world.

Briefly describe how RNA nucleotides can become protocells to support this hypothesis.

Answer 35

Initially, synthesis and RANDOM POLYMERISATION of nucletoides results in pools of nucleic acid oligomers, in which temple-directed non-enzymatic replication may occur.

RECOMBINATION reactions result in the generation of longer oligomers. Both long and short oligomers can fold into structures of varying complexity, resulting in EMERGENCE OF FUNCTIONAL RIBOZYMES.

ENCAPSULATION results in protocells with distinct genetic identities capable of evolution.

As complexity increases, the first RNA REPLICASE EMERGES.

Question 36

In onthophagus beetles, the transcription of horn development in determined by __________ hormone.

Answer 36

Juvenile Hormone (JH)

More JH (present in larger bodies); bigger horn

Question 37

In onthophagus beetles, males and females have unique doublesex isoforms. Large males ________ dsx compared to small males and females.

Male doublesex RNAi ______ large horns of large males and ________ ectopic horns in hornless females! Therefore:

Answer 37

Upregulate

Reduces; Induces

Therefore, doublesex has OPPOSING ROLES between males and females!

Question 38

What is the most common RNA edit? What other editing are there?

Answer 38

Most common: A to I

Other edit: C to U

Question 39

What does RNA editing in exons lead to? What does RNA editing in introns lead to?

Answer 39

Exons: different aa sequence or truncated protein

Introns: alter splicing, transport, efficiency of translation

Question 40

(T/F) RNA editing alters pre-mRNA sequences. While RNA editing is much widespread in protozoan, plant mitochondria and cholorplasts where there are additions and deletions of multiple bases, RNA editing in eukaryotes is much rarer and only single base changes are found.

Answer 40

True!

Question 41

What is the A-to-I editing done by in humans? How many genes have this editing?

Answer 41

A-to-I editing is done by ADARs (adenosine deaminase acting on RNA); ADAR1 and ADAR2.

There are 1,000 genes in humans with this edit.

Question 42

The liver cell has the unedited mRNA (CAA of exon 26) while the intestine cell has the edited mRNA (CAA –> UAA).

While both of the proteins’ N-terminal domain ASSOCIATES WITH LIPIDS, how do their C-terminal domain differ?

Answer 42

Liver cell C-terminal domain: BINDS TO LDL RECEPTORS on cell membranes - delivers cholesterol to all cells via receptor-mediated endocytosis.

Intestinal cell C-terminal domain: No C-terminal domain; fails to bind to LDL receptors.

Question 43

How do cold temperatures hinder neuron’s ability send signals?

Answer 43

When a nerve cell fires, protein channels in its membrane open or close to allow various ions in or out.

Frigid temperatures can delay the potassium channel’s closing, hindering the neuron’s ability to fire again.

Question 44

Antarctic octopus edits its RNA at ______ sites that change the amino acid sequence of the potassium channel.

One of these sites, known as _______, is important for adapting to the cold: the change more than _________ the potassium channel’s closing speed.

Answer 44

nine

I321V; doubles

Question 45

What is Ira?

What can defective NF1 cause?

Answer 45

Ira is a GTPase activating protein (GAP) that activates the GTPase activity of the Ras monomeric G protein that stimulates cell division, turning Ras off.

Defective NF1 could cause abnormally high Ras signaling leading to excessive cell division and formation of the tumors.