Quiz #2 (Module 2), Second 1/2 (Experimental + Techniques Part #2)

Spaces between exons

introns

Exons contain a _ in them

coding sequence (CDS)

Exons can also contain a _ in them

Untranslated regions (UTR’s), 5’ to 3” UTR

What will you do the genomic DNA?

transcribe it into an mRNA (taking a specific segment of DNA and copying it into a single stranded mRNA by the enzyme RNA polymerase

That transcribed mRNA will still have what similar components as the DNA

UTR’s (5’ to 3’), CDS

First codon of the CDS will always be

ATG

Genes are _ and many protein encoding genes are also _

transcribed; translated and a protein is produced

Why are genes expressed in such a specific pattern?

Key is the regulatory sequences that are outside of the coding of the gene (promotors)

Promotor

Part of the gene that is usually upstream, usually 5’ from the body of the gene. That contains regulatory regions.

What are the two types of regulatory regions?

Enhancers → promote transcription of the gene. Repressors → those sequences will inhibit the transcription of the gene in certain tissues

Enhancers and Repressors are

DNA sequences, places where other factors (like Transcription Factors (TF)) are going to bind.

Transcription factors

are the ones recruiting the main form of the RNA polymerase that is present express messages to things that are coding, that is RNA polymerase II and promote the transcription

The sequence that is upstream of the gene is usually the promoter and contains regulatory sequences, but that doesn’t mean they’re are not other _

regulatory sequences that may be located in the 3’ of the gene, could also have repressors located in the introns or 3’ part of the gene

So basically the complex regulation of where the gene is expressed is not necessarily encoded by the

coding sequence of the gene, but by the DNA that is surrounding the gene

We can modify the DNA to be able to look at gene expression using

reporters

Making a reporter

Create a transgenic line, manipulate the genome to be able to take the regulatory elements that control the expression of that particular gene. And put under the control of that regulatory region of a gene that i’m interested in, to be able to visualize it.

Technique to make copies of the promoter you’re interested in?

PCR (amplifies the region of the promotor sequence, making a copy of that specific DNA regulatory segment).

Instead of having the CDS for that specific gene that may encode for something were not interested in. Instead we will use the

CDS of a reporter (something im going to be able to detect)

Examples of a reporter

GFP (green fluorescent protein), RFP, YFP, CFP,

What else can we use as reporters that were originally used prior to discovery of FP’s (fluorescent proteins) ?

Enzymes; example) Beta-galactosidase

If you use an enzyme will it or will it not fluoresce?

It will not fluoresce, but you can use a substrate that potentially doesnt have any color, but will produce a product that will have a color, the example produces a precipitate that is blue (that region is called x-gal)

Other enzyme examples that could be used as a reporter

Luciferase (will take a substrate and produce something that is luminescent), AP

When we create a transgene

We are taking the gene of another species (ex: GFP comes from a jellyfish tree) and putting them inside the genome of your animal of interest. “Transfusing a new gene into the gene into the DNA of that species”.

Trans genes have three important components

1) Promotor → will tell where and when its expressed

2) CDS

3) Poly-A signal → promotor using those enhancers/repressors is attracting RNA polymerase II, it will bind here, start transcription, but needs to stop. To stop we will use a poly-A signal that tells RNA polymerase you should stop.

Example of a listed transgene

atoh1: B-gal-pA

Risks of transgenes

1) When putting into the genome it can land somewhere (completely random)

2) Certainty that the expression of the reporter is going to reproduce the expression of the gene absolutely faithfully? → wont be sure since only taking 5’ and amplifying, leaving 3’ regulatory element behind

Instead of creating an artificial transgene and jumping randomly into the genome →

You can modify the gene in this specific locus (homologous recombination → modifying the genomic DNA, preserving the entire structure of the gene)

Knock in

Replace the gene in the endogenous locus with the reporter, now actually has the 5’ and 3’ regulatory elements, using homologous recombination → do so by inserting anything that I want in this specific locus modifying the genomic DNA, preserving the entire structure of the gene

“put it in that particular space”

Move it

gain of functions

Pax3 mutation

causes no iris to be present in eyes (eye disease)

In many species, in the case of Drosophila, genes are named first because

of the phenotype that they produce

Forward genetics

Two main key words when working with genes in a move it experiment

If a gene is necessary and if it is sufficient

Pax3 and eyeless mutant (ey) are the

same thing, just an overexpression of the gene in a different area than it normally is

Pax3 is a _ factor that is extremely conserved called _ in drosophila

transcription; eyeless (ey)

Why did the fly have eyes in their limb?

Produced by overexpressing, by moving the gene (ey) or (Pax3) under the control of different promotors

Binary system is

when you have one transgene that is driving the expression of this transcription factor (GAL4 for the example) and then the resulting expression (eyeless in this example).

Foward genetics screen

1) Decide that you’re interested in a specific phenotype

2) Saturate the genome with mutations, try to mutate every gene in the genome (mutagenesis)

3) Try to recover phenotypes that have the phenotype that i’m interested in

Chemical mutagenesis

Using drugs that are going to affect specifically the DNA, want to induce random mutations. Mutations are not specifically located in any gene, any developmental gene, just random mutations.

Other types of mutagenesis

radiation and insertional

Benefit of chemical mutagenesis compared to radiation

induces single nucleotide changes, while radiation tend to create large mutations, large deletions, and also complex rearrangements

positional cloning linkage analysis

An incredibly painful process. Have a mutant and phenotype, but no idea of what specific gene is actually mutated.

All transgenic lines are essentially what type of experiment?

Find it - if they are reporters

Reverse genetics

1) Take one gene and target it, mutate that gene

2) observing the resulting phenotype

If we are inducing a mutation specifically that is

reverse genetics

Reverse genetics target mutagenesis

CRISPR/Cas9

Cas9 is a

nuclease (an enzyme) that will cut the DNA, will induce double strand breaks in the DNA, but does not have specificity (like restriction enzymes do). Relies on the specificity of a guide RNA (gRNA)

Cas9 and the gRNA will couple then

they will scan the DNA, find where to cut and induce double strand breaks

Once the double strand break occurs

it will rigger an alarm system for the cell. Have to repair, but not perfectly. Repair will either lose or add nucleotides around the breaking point (insertion vs deletion).

Indels

are those insertions and deletions of nucleotides in a DNA sequence

These deletions and insertions of one nucleotide can cause

reading frame to change (deletion -), insertion of (+2) causes a stop codon, insertion of (+3) could get lucky and have no disruption at all

Lineage tracing

What is the source of one particular structure or what is actually the contributions of some particular cell types

Lineage tracing first done with

pigmentation of the embryos, follow lineage of pigmented blastomere. pigment will stay throughout gastrulation and further.

Fate Mapping

Taking pieces of carbon and staining different areas of the embryo and then looking at the contributions of those particular areas. Can do it with carbon, some dyes that have physical color, or with fluorescent dyes. What are the fate of those cells?

Lineage tracing by Transplantation (Xenograph)

Interspecies transplantations → qual embryos as donors and chicken embryos as host → take piece of quail embryo and transplant into chick embryo → later recognize the quail embryos (cells), because they express a protein called QCPNA, will always express this protein so can track → detect with an antibody → not imunnofluorescent, but immunohistochemistry (reaction where some cells in the neuro tube are derived from the quail)

Fluorescent dyes have what problem with lineage tracing

They will fluoresce for a while, but eventually dilute, demetabolize.

Transplantation of cells from an embryo that is differentially labeled with fluorescent proteins into another

one that is not labeled more common, follow lineage later on (ex) GFP in the host cells, still in nueral tube, but later on nueral crest cells migrate down (find it experiment)

Why do the fluorescent proteins transplanted from the embryo last longer?

Continuance production of GFP is what differs here

Why are all forms of lineage tracing a little bit of a trap?

Fluorescent dyes will dilute and not be seen in adult. Transplantation of another species is also a trap → Immune system will kick in and reject cells from other species (without medicine wont stand). Even if cells from same species → still cells from different individuals.

Therefore anyway to correctly do lineage tracing is to do

genetic lineage tracing