Dev.

Bio:

Development is the process of...

progressive and continuous change that generates a complex
multicellular organism from a single cell.

Untranslated regions are _____ but not ______.

transcribed, translated.

Is the TATA box found in all genes?

No.

Intronic sequences are spliced out of the...

pre-mRNA.

The intron is removed for formation of a...

spliceosome loop structure.

T or F: Alternative RNA splicing greatly increases the number of

protein products possible from each gene.
True.

Myostatin is...
a key inhibitor of myogenic gene networks.

A mutation in myostatin creates...

a new splice acceptor site that leads to a mutated protein.

AG is the splice...
acceptor.

A splicing mutation has the same...

phenotype as a null mutation in myostatin.

GT is the splice...
donor.

What does an enhancer region do?

It controls tissue-specific gene expression.

Mutations in the Duffy protein regulatory element increase what?

Resistance to malaria.

What regulates gene expression levels?

microRNAs.

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microRNAs can be encoded in...

stand-alone genes or within introns of protein coding genes.

What is RISC and whats does it do?

RISC is the RNA Induced Silencing Complex and it destroys
double-stranded RNA.

What are the 3 ways that the miRNA complex can block
translation?
1. Initiation block
2. Endonuclease digestion
3. Proteolysis

Gene regulation refers to the cellular processes that...

control the rate and manner of gene expression.

What is the most economical method of regulation?

The rate of transcription.

What assay uses anti-sense RNA probes?

In Situ Hybridization.

In situ hybridization provides temporal and spatial information

about gene expression by detecting...
mRNA.

Is in situ hyridization quantitative or qualitative?

Qualitative. It doesn't tell you how much, just that it's there.

RT-PCR allows us to look at genes expressed in...

tissues and cells.

With RT-PCR what can you isolate from the sample?

RNA.

Is RT-PCR quantitative or qualitative?

Quantitative.

A DNA microarray is an orderly arrangement of...

DNA probes on a slide.

What is DNA microarray good for?

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Comparing two samples and looking at global expression.

T or F: You must know the gene identity of each probe to use a

microarray.
True.

Is a DNA Microarray quantitative or qualitative?

Quantitative.

Microarrays allow for rapid identification of many genes that are...

altered in their expression pattern in a particular situation.

Comparison using microarrays allows scientists to what?

Get information regarding the effect of a disease or mutation on
other genes.

How does altering gene expression help us?

It provides information on the function of a gene, genes it regulates
and its relative importance to development of specific organs and
tissues.

What array silences the expression of specific genes?

RNA interference.

RNA interference allows examination of the role or function of a

gene product WITHOUT...
the need to make a genomic mutation.

If you found that actin is highly expressed in normal cells but not
in cancer cells, what would you conclude?
Cancerous cells turn off actin expression.

RNA interference is a mechanism for silencing a gene expression

by acting on...
mRNA stability and mRNA translation.

What is molecular cloning?

Molecular biological methods that are used to assemble
recombinant DNA molecules and to direct their replication within a
host organism.

What is a plasmid?
A circular double-stranded extrachromosomal DNA molecule.

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What is a plasmid used for?

To clone, transfer, and manipulate genes-once DNA fragments or
genes inserted into them.

What is homologous recombination?

A type of genetic recombination in which nucleotide sequences are
exchanged between two similar or identical molecules of DNA.

How can DNA integrate into chromosomes nonhomologously?

By end-joining at double strand breaks. Requires linear dsDNA with
free ends.

To make knockouts we use...

homologous recombination, for gene targeting.

To make transgenes we use...

non-homologous end joining, random integration of DNA.

What do you need to make a transgenic mouse?

Targeting vector, fertilized oocytes, psuedopregnant foster mother,
method to screen for targeted embryos or pups.

What is a transgene?
An artificial gene, must incorporate all appropriate elements critical
for gene expression.

What does a transgene contain?

A promoter, an intron, a protein coding sequence (the reporter), and
transcriptional stop sequence.

What can you learn from a transgenic mouse?

1. Where are tissue/cell specific enhancers
2. When during embryonic time an enhancer is functional
3. What does a specific cell lineage become
4. Where do cells of a specific lineage migrate

What is an Embryonic Stem Cell?

A pluripotent cell, derived from inner cell mass of blastocyst.

To make a transgene that expresses gene X you must clone it

into...
a plasmid.

A transgene embryo is in what cells?

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Only fertilized eggs.

You are making a transgene, where do you predict it will integrate

into the genome?
A random chromosomal location. When making a transgene it
doesn't matter where it lands, it's random integration.

Embryonic stem cells are crucial to making knock-out mice

because they are...
Pluripotent: ESC can't make membranes so it can't create an
entirely new cell.

Induction is the process...

by which one group of cells changes the development of an
adjacent set of cells.

What are the two types of cells in induction?

The inducer and the responder.

The inducer is the tissue that...

produces a signal that can change the behavior of the other tissue.

The responder is the tissue that...

responds to inducing signal.

A cell's ability to respond is referred to as...

competence.

Instructive Induction is...

when there is no instruction by the Inducer, the Responder cell
cannot follow a certain cell fate. EX: a coach telling individual
football players what to do.

Permissive Induction is...

when the Responder cell has already been specified in cell fate, but
needs a generic cue from the inducer.

Reciprocal induction is...

observed when both communicating cells can serve as inducer and
responder.

An example of induction is...

feathers on forelimbs vs. scales on hindlimbs of chickens. The
inducer tells the cells what to develop.

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Where does the specificity of induction lie?

Within the mesenchyme.

Proximal is...
closer to the center of the body. EX: the elbow is more proximal
than the wrist.

Distal is...
more distant from the center of the body. EX: the wrist is more distal
that the elbow.

Rostral is...
towards the head. EX: the shoulder is more rostral than the elbow.

Caudal is...
towards the tail. EX: the elbow is more caudal than the shoulder.

Dorsal is...
the upper surface. EX: top side of your hand.

The thumb is always more _____, than the pinky.

rostral

Ventral is...
the underside. EX: the palm of your hand.

In animals anterior and posterior are used to describe....

rostral and caudal.

What is the skeletal anatomy of the chick upper extremity?

Humerus, ulna, radius, metacarpals, digits.

The AER (apical ectodermal ridge) is a key inducer of...

proximal-distal outgrowth.

The AER is required for what?

Limb bud outgrowth.

Removing the AER does what?

Halts distal outgrowth of the limb.

The later in limb bud development you remove the AER....

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the more distal structures are formed.

What is the inductive factor in the AER?

Fgf8

What does the AER induce?

The underlying mesenchyme.

Leg mesenchyme is inserted just below the AER in the forelimb,

which tissue decides the type of limb formed-the AER or the
underlying mesenchyme?
The mesenchyme, which is what forms the outgrowing limb.

Non-limb AER mesenchyme is placed under the AER. Why can't

this be induced to form limb tissue?
The non-limb mesenchyme is not competent to be induced.

What is the Zone of Polarizing Activity (ZPA)?

The Caudal/Posterior Signaling center in the Limb Bud.

Sonic hedgehog (SHH) protein is morphogen expressed in...

the ZPA.

What are morphogens?

diffusible molecules that are recognized by cells.

An embryonic field is defined by the prescence of a morphogen

_____ and a ____, creating a gradient morphogen.
source, sink.

What is a morphogen source?

The site of synthesis.

What is a morphogen sink?

The site of degradation.

What alters the slope of the gradient?

the rate of synthesis and degradation.

Ectopic expression of SHH in the rostral/anterior limb bud

causes....
polydactyly.

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If you take mesenchyme form the foot and put it under the thigh,
what would you expect to see?
Scales from the foot.

A developmental biologist transplanted some forelimb bud

mesenchyme underneath the AER of a hindlimb bud mid-way
through limb bud growth. What would you expect to see?
An appendage that is proximal leg and distal wing.

Why do you want to have sexual reproduction?

to diversify.

Out of the 3 inductions, which one is almost always happening?

STOPPED!!!!!
Reciprocal.

Duplication of the ZPA results in...

mirror image duplication along the rostral-caudal axis.

T or F: Animal cells can be disaggregated.

True.

By having preferential adhesion, cells can...

aggregate with similar cells.

Differential adhesion is based on...

selective cell-cell interactions mediated by binding of cell surface
receptors.

In differential adhesion cell types with the highest affinity will be

surrounded by cells with...
a weaker affinity.

Cadherin helps..
mediate differential adhesion.

Cadherin is required for...

cell adhesion and embryonic development.

Juxtacrine signaling is when...

cells membranes are touching.

Notch signaling is...

protease resistant.

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Paracrine signaling is when...

cells are not touching but close. A diffusible factor carries signal.

What is happening molecularly during paracrine signaling?

1. Inducing cells influence their neighbors by secreting small
proteins that diffuse to responding cells and bind to membrane
receptors.
2. initiates an intracellular signaling cascade through a series of
signal transduction proteins.
3. these proteins enter the nucleus, where they alter gene
expression (transcription), leading to the synthesis of new proteins.

Morphogen gradients are formed from...

paracrine signals.

The extracellular matrix secreted by cells can...

send signals to other cells.

Signals from the extracellular matrix can...

mediate permissive induction.

What are integrins?

heterodimeric receptors that consist of alpha and beta subunits.
Beta binds to cytoskeletal elements, alpha binds to signal
transduction proteins.

The cytoskeleton of a cell can be linked to the extracellular matrix

through...
integrin attachments.

What is the mechanism in notch signaling by which the inducing

cells changes target gene expression in the responding cell?
Transcriptional regulation.

In a chick embryo, if you remove the AER early, just after

development of the forelimb bud, what would you expect to see in
the hatched chick?
A wing that is truncated after the middle of the humerus.

The optic vesicle of a frog embryo is placed in a different part of

the head ectoderm, it will induce that ectoderm to form lens
tissue. However, if the optic vesicle is placed beneath ectoderm in

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the trunk, that ectoderm will not form lens tissue. What describes
the interaction of these tissues?
The head ectoderm is competent to respond to signals from the
optic vesicle while the trunk ectoderm is not.

What is expressed in the ZPA that produces a rostral-caudal

morphogen gradient in the limb bud?
SHH.

You are growing two types of cells, mesenchyme and epidermal,

in two different dishes. If you take the liquid medium only, no cells,
from the mesenchymal dish and place some of it into the
epidermal dish, the epidermal cells begin to proliferate and
change shape. What type of cell-cell interaction is taking place?
Paracrine signaling.

The delta ligand on one cell binding to the notch receptor of

another cell is an example of what?
Juxtracrine signaling.

A mutant mouse lacks the SHH gene will have limbs with
disrupted _____ specification of the forelimb.
Caudal.

For notch signaling, notch binds to...

the adjacent cell, receives signal, breaks off and binds to the
repressor, transcription occurs.

What are the 2 Embryonic Stem Cell selections?

Positive and Negative.

What is positive selection?

Neomycin resistance makes cells resistant to the antibiotic G418 or
geneticin.

What is negative selection?

Ganciclovir acts a prodrug, it is not toxic, but is converted to a toxin
by phosphorylation by viral thymidine kinase.

What happens when cells express viral TK (thymidine kinase)?

it produces highly toxic triphosphates that lead to cell death.

Why do ES cells need to contribute to the germline in creating a

knockout mouse?

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They won't be able to breed the mice with the targeted mutation
without contributing.

When blastocysts are injected into the uterus of psuedopregnant

mothers you then allow mice...
to develop and look at the genotype of pups. Then back cross onto
wild type to find those mice that carry trait in the germite.

What does knock-in mean?

It's another way to examine gene expression by "knocking-in" a
reporter into the normal locus of the gene of interest.

Are the procedures of knock-outs and knock-ins the same?

Yes.

Cre-recombinase is....
a bacterial enzyme that recognizes a distinct sequence in DNA loxP site.

Cre-recombinase can be used to make...

site specific gene lesions- both in a specific time and place.

Cre-recombinase requires two separate mouse lines:

1. expressed Cre recombinase in some fashion (often a transgene)
- this is Cre line
2. has a floxed allele; is flanked by loxP sites- this is the conditional
allele.

How do you generate a floxed allele?

Clone a LoxP site around the sequence that Cre recombinase is to
remove targeting vector. When Cre recombinase is present it binds
at LoxP sites. Cre recombinase makes a double strand break and
repairs it leaving behind one LoxP site and removing the intervening
DNA.

What is Fgf8?
A soluble signaling factor that is expressed in developing limbs.

A traditional knock out of Fgf8 resulted in...

an early embryonic lethal mutation.

How do you look at the role of Fgf8?

Breed the Fgf8 floxed allele with a mouse line that expressed Cre
recombinase under the control of a promoter that is expressed only

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in the developing forelimb. In situ hybridization with an Fgf8 probe

shows reduced expression of the AER of developing limbs.

The Cre/Lox approach demonstrated that Fgf8 is...

necessary for limb outgrowth and patterning.

If you have targeted ES cells and inject them into a blastocyst...

when you inject you put a selection of ES cells in to mix with
already present cells, the cells that are already there aren't targeted
and that's why it's some embryonic cells will have the targeted
gene.

What are the key attributes of ES cells for experimental

development?
They are pluripotent and can be manipulated.

A mouse with a floxed Fgf8 allele was bred to a mouse with a Cre
recombinase transgene that is expressed only in the forelimb, not
the hindlimb, then...
The cells of the forelimbs will have recombined Fgf8.

If you have a mouse line that expressed cre recombinase under

the control of a heart specific enhancer, what is true?
Only cells in the heart express cre recombinase.

Some cells, the targeting vector recombines with the target gene
and knocks out one copy of the target gene, which is...
neomycin-resistant and ganciclovin-resistant

Other cells the targeting vector recombines in the wrong place, a

random section of the chromosome, which is...
neomycin-resistant and ganciclovir-sensitive.

How many generations does it take to breed out a homozygous

animal?
Two.

The process leading to cell differentiation:

1. Commitment - the cell's developmental fate has been restricted
even though this is not overtly displayed by a change in functional
or morphological features.
2. Differentiation - the generation of specialized cell types from
committed precursors.

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Commitment is made up of two steps:

1A. Specification - still reversible
2A. Determination - irreversible

Two types of specification:

Autonomous specification - cells achieve their fates by factors
received from their parental cells, irrespective of surrounding cells.
Conditional specification - cells achieve their fates by interactions
with other cells, (cell-cell communication).

The nematode Caenorhabditis elegans:

is a model of autonomous and conditional specifications.

C. Elegans embryos differ from mammalian or bird embryos in

that their...
embryonic cell divisions are invariant, they always divide into the
same daughter cells in the same locations.

C. elegans embryo polarity is established by...

sperm entry and spindle orientation.

An example of autonomous specification in C. elegans:

P-granule segregation into C. elegans germline lineage.

C. elegans also has an example for what type of signaling?

Juxtacrine notch signaling.

Wnt7a is required for...

dorsal development. This suggests that ventral development is the
default state.

A Wnt7a mutant is...

ventralized transformation of the limb due to a loss of dorsalizing
factor.

Wnt7a is expressed in the...

dorsal limb bud ectoderm, specifying dorsal fate.

The following is a diagram from Ensembl of the

MC1R gene that regulates red hair color.
Where is the translational start site?

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A is the transcriptional start site

Diagram of a protein coding gene

The following is a diagram from Ensembl of the

MC1R gene that regulates red hair color. Which
of the following represents the 5' UTR?

Diagram of a protein coding gene

(untranslated regions)

What is the genomic signal that tells the cell to

start transcription?
the promoter sequences are required to initiate
transcription

Enhancers
they are the sites of transcription factor binding.
Have Flexibility in Their Orientation and
Localization
Tissue Specific and Modular

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In individuals with the Duffy mutation that

increases resistance to malaria, what level of
expression to you expect for the Duffy protein in
the endothelial and Purkinje (brain) cells?
normal; enhancers are tissue-specific. That
means that the effects on the transcription of that
gene are specific to the tissue controlled by that
enhancer element. Mutation of the blood cell
enhancer does not affect other tissues.

Key signals for a protein coding gene

Promoter region and PolyA addition site

In the diagram below, what are the nucleotides

shown in the boxes?
Binding sites for the spliceosome factors

What does myostatin do?

inhibits myogenesis

You want to study the process of development

in a relatively unstudied species, the green
anole lizard. The following is the best method
for quantifying expression of as many genes as
possible in specific parts of the embryo?
RNA-Seq, assays all genes in genome

Development
The process of progressive and continuous
change that generates a complex multicellular
organism from a single cell.

Eukaryotic promoter structure

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TATA Box (Goldberg-Hogness box)

A/T rich region located ~ 30 bp
The consensus sequence is
TATA(A/T)A(A/T)
Not found in all genes. Notably absent in some
housekeeping genes that are expressed at in all
cells.

What do transcription factors do?

Bind to Enhancers and Form a Bridge to Activate
Transcription from the Promoter

Enhancer characteristics
Have Flexibility in Their Orientation and
Localization
Tissue Specific and Modular
regions control tissue-specific gene expression

tissue-specific gene expression

Mutations in the Duffy protein enhancers

found in lowland area populations and increases
resistance to malaria

What happens to Intronic sequences?

removed for formation of a spliceosome loop
structure. Sequences are spliced out of the premRNA

A mutation in myostatin creates a new splice

acceptor site that leads to a mutated protein
A splicing mutation has the same phenotype as a
null mutation in myostatin.

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A plasmid is?
Circular extrachromosomal DNA

A plasmid vector contains three elements

- Cloning site where the foreign DNA fragment
can be inserted
- Drug resistant gene that destroys antibiotics to
allow selected growth for the host cell
- Replication origin to allow the plasmid to
replicate in the host cell.

What is Transformation?
A process that involves a few cells taking in the
recombinant plasmid. Most cells don't and
eventually die when placed in the antibiotic
infused nutrient medium.

Homologous recombination
Nucleotide sequences are exchanged between
two similar or identical molecules of DNA.

Nonhomologous recombination (random

integration)
DNA can integrate into chromosomes by endjoining at double strand breaks. Requires linear
dsDNA with free ends.

TATA Box (Goldberg-Hogness box)

A/T rich region located ~ 30 bp
The consensus sequence is TATA(A/T)A(A/T)
Not found in all genes. Notably absent in some
housekeeping genes that are expressed at in all
cells.

Experimental Methods to Assay Gene

Expression
Spatial & Quantitative

Spatial method
- In situ hybridization
- Transgenic reporter gene

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Quantitative method
- Quantitative RT-PCR
- RNA-Seq

In situ hybridization with anti-sense RNA probes

- Embryo must be fixed before analysis.
- Only 1-2 genes examined at a time.
- Not quantitative

Transgenic Reporter Genes

A DNA construct can be made fusing the
promoter and enhancers of the gene of interest
with a fluorescence or enzyme reporter gene.
This is then inserted into the somatic genome.

Reverse transcription polymerase chain

reaction (RT-PCR)
RT-PCR allows us to look at genes expressed in
tissues and cells.
Snapshot of expression of many genes, but you
have to destroy the cells/tissue to extract RNA
from the sample.
Quantitative RT-PCR provides measure of the
expression for one gene per assay.
Multiple assays can be run at a time, up to
hundreds.

RNA-Seq (RNA Sequencing)

- Embryo must be destroyed for analysis.
- All genes can be asayed, even ones you do not
know about.
- The most sensitive and quantitatively accurate
assay with current technology.

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Gene Expression Methods Diagram

Identify the key steps and elements required for

transcription and processing of mRNA,
including but not limited to the role of the
promoter and splicing
...

Recognize the role of differential gene

transcription in development:
transcription factors and enhancers
tissue specificity of enhancers
Griffiths, 9th Ed., Ch. 8

Describe how selective nuclear RNA

processing can generate multiple isoforms by
alternate splicing
...

Compare and contrast each methods to assay

gene expression, including in situ hybridization,
reporter genes, quantitative RT-PCR, and RNASeq. Recall which methods yield spatial vs.
quantitative data.
...

Modern molecular genetics revolutionized the

study of vertebrate development
1982: Richard Palmiter and Ralph Brinster create
the first transgenic mouse.

Making a transgene
- artificial gene; must incorporate elements
critical for gene expression.
- A simple construction scheme has been
developed that provides the best transgene

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expression: each transgene contains a promoter,

an intron, a protein coding sequence,
transcriptional stop sequence and 3' polyA site.

Example transgene diagram

To make a transgene that expresses gene X

you must clone it into
a plasmid

What do you need to make a transgenic

mouse?
- Transgene
- Fertilized oocytes
- Pseudopregnant foster mother
- Method to screen for targeted embryos or pups

where are transgenes located when

implemented?
all cells of an embryo.

You are making a transgene, where do you

predict it will integrate into the genome?
A random chromosomal location

What can you learn from a transgenic mouse?

- Where are tissue/cell specific enhancers.
- When during embryonic time an enhancer is
functional.
- What does a specific cell lineage become.
- Where do cells of a specific lineage migrate.
Role of specific genes in development.

Where are tissue specific enhancers for a

specific gene?
in the enhancers for that particular tissue

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When/where is a gene expressed during

embryogenesis.

Why make a knock out mouse?

allows us to ask important questions about the
function of gene products.
learn what the role of a specific gene product is
by studying development in its absence
huge leap forward for mammalian developmental
biology

What do you need to make a knock-out?

- Embryonic stem cells
- Sequence and structure of the gene locus
- Targeting vector
- Method to screen for targeted ES cells
- Blastocysts
- Pseudopregnant foster mother
- Method to detect chimeric neonates

Pluripotent
stem cells; can become all of embryo, but not
extraembryonic tissues (eg. placenta,
membranes)

Embryonic stem cells (ESCs)

Pluripotent stem cells- can become all of embryo,
but not extraembryonic tissues (eg. placenta,
membranes)

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From the inner cell mass of the blastocyst

Can culture indefinitely in undifferentiated state
Discovery of viable culture conditions of mESCs
led to knock out/knock in and transgenic genetic
manipulations of mouse embryos
1998- James Thompson Univ. of Wisconsin
isolated and cultured human ESCs for the first
time

Homologous recombination
a type of genetic recombination also known as
crossing over that occurs during meiosis.
It also occurs in diploid cells under special
circumstances, this is important for ES cell
mediated knockouts.
Gene map translated parts are represented by
shaded spots

Gene map parts that are untranslated are represented by

unshaded parts

Gene map introns are represented by

lines

Gene map exons are represented by

Boxes

Transcription is started by
a promoter (tata box)

Translation is started by
AUG start codon

what is the purpose if the 3' UTR

stability, protects against degradation, used by microRNA

induction
the process by which one group of cells changes the development
of an adjacent set of cells (shape, mitotic rate, cell fate)

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what are the 2 types of cells in induction

inducer and responder

what is an inducer
tissue that produces a signal and can change behavior of other
tissue

what is a responder
tissue that responds to inducing signal, must be competent to
respond

instructive induction
coach telling players what to do -> inducer tells responder what to
do

committment
cells developmental fate has been restricted even though this is not
overtly displayed

differentiation
generation of specialized cell types from committed precursors

steps of committment:
1. specification: still reversible
2. determination: irreversible

Proximal-distal inducing factor

FGF8 in the AER

Rostral-caudal inducing factor

SHH in the ZPA

Dorsal-ventral inducing factor

Wnt7a in dorsal surface (produces dorsal growth, absence =
ventral)

hox genes
specify regions along limb bud in proximal-distal axis
The phrase "all the somatic cells in a developing embryo contain
the same set of genes" refers to
Genomic equivalence

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_______ ________ examines how gene expression is regulated

to coordinate embryo development
Developmental genetics (regulation of the processes in the central
dogma control gene expression)

a ______ binds RNA polymerase and initiates transcription

Promoter

Promoter
-Regulate when and where a gene is transcribed
-Promoters can be found at the 5' end; 3' end; or within gene
-Usually contains a sequence called the TATA box

_________ ______ _____ is a "cap sequence" where RNA

polymerase starts making the RNA transcript
Transcription Initiation Site

What are the characteristic of a transcription initiation site?

-Generates the 5' end of the mRN where the Guanosine-Cap (Gcap) is added
-G-Cap is required for binding to the ribosome before translation
-Different genes have different sequences at their transcription
ignition site

The __________ _________ _____ is where the translation of

RNA to peptide will begin
Translation Initiation Codon

the 5' region of DNA btw the transcription ignition sequence and
the translation sequence where the mRNA is to translated, but
can determine the rate that translation is initiated is referred to
as?
5' UTR

The region of the mRNA that codes for proteins is called?

Exon

The intervening regions that do not code for proteins, but for
micro RNAs is called?
Intron

The site where transcription stops is called?

Transcription termination site

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the region of the RNA transcript is NOT translated is known as?

3' UTR region

What is the region on a gene that is used to generate the Poly-A

tail (adenosine are added enzymatically and are not coded by the
DNA sequence)?
3' UTR

What are the three functions of the Poly-A tail?

1) stabilizes the mRNA
2) facilitates transport from the nucleus
3) allows translation of the mRNA

Describe the four factors that regulate gene expression?

1) Differential Gene Expression:
Accessibility to DNA, promoter activity, enhancer/silencer
sequences, transcription factors, methylation
2) Differential RNA Processing: RNA selection regulates the nRNA
that enter the cytoplasm to become mRNA
RNA splicing generates multiple proteins from a single mRNA
3) Regulation of Translation: microRNAs, localization of mRNAs
4) Post-Translational Modifications: regulates protein function within
cell

How does histones regulate gene expression?

Histones act as an "activation switch" regulating gene expression
by controlling what genes are available for transcription

What is the difference btw condensed and uncondensed

nucleosomes?
Condensed nucleosomes have histone tails that are methylated
and uncondensed nucleosomes have histone tails that are
unmethylated and acetylated 1

How does condensed chromatin repress gene expression?

prevent access to transcription machinery

How does uncondensed chromatin activate gene expression?

allow access to transcription machinery

What is the difference btw histone acetylation and histone deacettylation in terms of gene expression?
Histone acetylation activates transcription, whereas histone deacetylation repress transcription

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_______ are regulatory sequences found in the 5' UTR; 3' UTR;
or introns of a gene
Enhancers

What is the function of an enhancer?

regulate when a promoter is active and how much is transcribed;
bind specific combinations of transcription factors to activate
transcription

What is enhancer modularity?

Each enhancer controls expression independently, at different
times/places, direct "tissue specific" gene expression

What is enhancer mechanism?

when enhancers bind transcription factors that recruit additional
accessory factors (HDACs, RNA polymerase) to activate
transcription

A enhancer can?
bind several different transcription factors ti activate a particular
gene
bind different combinations of transcription factors which controls
the timing and location of differ gene expression patterns

________ are DNA regulatory elements that repress gene

transcription?
Silencers

Which type of silencer only allows promoter activation in

neurons?
NSRE (Neural Restrictive Silencer Element)

How does NSRE block transcription?

binds the transcription factor NRSF (neural restrictive silencer
factor)

_________ is the study of heritable changes in gene expression

or cellular phenotype by mechanism other than changes in the
DNA sequence
Epigenetic

What are the ways DNA methylation can block transcription?

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1) Methylation of cytosine, section of DNA becomes inactive

2) Methylated cytosine residues can recruit proteins that stabilize
the nucleosome and restrict access to the DNA: recruit histone
deacetylases (HDACs) that remove acutely groups or recruit
histone methyltransferases that add methyl groups

What is the difference btw Dnmt3 (de novo mtheyltransferase)

and Dnmt1 (perpetuating methyltransferase)?
Dnmt3: adds methyl groups to unmethylated cytosine residues
Dnmt1: recognizes methylcytosine on the parental strand and adds
a methyl group to the corresponding cytosine residue on the newly
synthesized strand

Inheritance of methylation patterns is important to?

ensure that gene expression remains stable

What is genomic imprinting, and how is it maintained?

Where methylation permanently regulates expression of either the
maternal or paternal allele of particular genes at the start of
development. (X chromosome methylation)

_______ _______ a phenomenon in mammals where only the

sperm derived or egg-derived allele is expressed?
Genomic Imprinting

How does DNA methylation ensure women are mosaic?

in early development, both X's are active, then 1 becomes inactive
via promoter methylation, inactivation is random and irreversible

What is RNA selection?

different cells will release different subsets of mRNA into the
cytoplasm to be processed, the same "pool" of nuclear RNA
transcripts will result in different cytoplasmic mRNA populations

When one mRNA transcript can produce several different proteins

is referred to as?
RNA splicing?

What are small RNA fragments that are complementary to mRNA

sequences and are non-coding genes that code for pre-mRNA?
MicroRNAs

How does microRNAs regulate translation?

regulate gene expression by blocking translation of selective mRNA

Dev. Bio:

Drosha (RNAase)
cleaves the pre-miRNA into single hairpin loops

Dicer (RNAase and Helicase)

cleaves non-base-paired domains and separate double-stranded
miRNA

How does microRNAs repress translation?

when binding of the miRNA and RNA is less complementary the
miRNA/RISC complex represses translation

When bindinfof the miRNA and mRNA is very tight, the mRNA is?
cleaved and degraded

What are the ways miRNAs block translation?

1) Initiation complex: prevent initiation complex from forming
2) Endonuclease digestion (de-adenylation): digests polyA tail
3) Proteolysis: facilitate protein degradation

What is the protector protein complex?

Binds to mRNA to facilitate translation

What is the most common method of localization of mRNAs?

mRNAs bound by their 3' UTR motor proteins are transported to
specific domains in the cytoplasm

What are the four post translational proteins modifications?

Phosphorylation
Glycosylation
Ubiquitination
S-Nitrosylation
Methylation
N-Acetylation
Lipidation
Proteolysis
The following is a diagram from Ensembl of the
MC1R gene that regulates red hair color.
Where is the translational start site?
A is the transcriptional start site

Dev. Bio:

Diagram of a protein coding gene

The following is a diagram from Ensembl of the

MC1R gene that regulates red hair color. Which
of the following represents the 5' UTR?

Diagram of a protein coding gene

(untranslated regions)

What is the genomic signal that tells the cell to

start transcription?
the promoter sequences are required to initiate
transcription

Enhancers
they are the sites of transcription factor binding.
Have Flexibility in Their Orientation and
Localization
Tissue Specific and Modular

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In individuals with the Duffy mutation that

increases resistance to malaria, what level of
expression to you expect for the Duffy protein in
the endothelial and Purkinje (brain) cells?
normal; enhancers are tissue-specific. That
means that the effects on the transcription of that
gene are specific to the tissue controlled by that
enhancer element. Mutation of the blood cell
enhancer does not affect other tissues.

Key signals for a protein coding gene

Promoter region and PolyA addition site

In the diagram below, what are the nucleotides

shown in the boxes?
Binding sites for the spliceosome factors

What does myostatin do?

inhibits myogenesis

You want to study the process of development

in a relatively unstudied species, the green
anole lizard. The following is the best method
for quantifying expression of as many genes as
possible in specific parts of the embryo?
RNA-Seq, assays all genes in genome

Development
The process of progressive and continuous
change that generates a complex multicellular
organism from a single cell.

Eukaryotic promoter structure

Dev. Bio:

TATA Box (Goldberg-Hogness box)

A/T rich region located ~ 30 bp
The consensus sequence is
TATA(A/T)A(A/T)
Not found in all genes. Notably absent in some
housekeeping genes that are expressed at in all
cells.

What do transcription factors do?

Bind to Enhancers and Form a Bridge to Activate
Transcription from the Promoter

Enhancer characteristics
Have Flexibility in Their Orientation and
Localization
Tissue Specific and Modular
regions control tissue-specific gene expression

tissue-specific gene expression

Mutations in the Duffy protein enhancers

found in lowland area populations and increases
resistance to malaria

What happens to Intronic sequences?

removed for formation of a spliceosome loop
structure. Sequences are spliced out of the premRNA

A mutation in myostatin creates a new splice

acceptor site that leads to a mutated protein
A splicing mutation has the same phenotype as a
null mutation in myostatin.

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A plasmid is?
Circular extrachromosomal DNA

A plasmid vector contains three elements

- Cloning site where the foreign DNA fragment
can be inserted
- Drug resistant gene that destroys antibiotics to
allow selected growth for the host cell
- Replication origin to allow the plasmid to
replicate in the host cell.

What is Transformation?
A process that involves a few cells taking in the
recombinant plasmid. Most cells don't and
eventually die when placed in the antibiotic
infused nutrient medium.

Homologous recombination
Nucleotide sequences are exchanged between
two similar or identical molecules of DNA.

Nonhomologous recombination (random

integration)
DNA can integrate into chromosomes by endjoining at double strand breaks. Requires linear
dsDNA with free ends.

TATA Box (Goldberg-Hogness box)

A/T rich region located ~ 30 bp
The consensus sequence is TATA(A/T)A(A/T)
Not found in all genes. Notably absent in some
housekeeping genes that are expressed at in all
cells.

Experimental Methods to Assay Gene

Expression
Spatial & Quantitative

Spatial method
- In situ hybridization
- Transgenic reporter gene

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Quantitative method
- Quantitative RT-PCR
- RNA-Seq

In situ hybridization with anti-sense RNA probes

- Embryo must be fixed before analysis.
- Only 1-2 genes examined at a time.
- Not quantitative

Transgenic Reporter Genes

A DNA construct can be made fusing the
promoter and enhancers of the gene of interest
with a fluorescence or enzyme reporter gene.
This is then inserted into the somatic genome.

Reverse transcription polymerase chain

reaction (RT-PCR)
RT-PCR allows us to look at genes expressed in
tissues and cells.
Snapshot of expression of many genes, but you
have to destroy the cells/tissue to extract RNA
from the sample.
Quantitative RT-PCR provides measure of the
expression for one gene per assay.
Multiple assays can be run at a time, up to
hundreds.

RNA-Seq (RNA Sequencing)

- Embryo must be destroyed for analysis.
- All genes can be asayed, even ones you do not
know about.
- The most sensitive and quantitatively accurate
assay with current technology.

Dev. Bio:

Gene Expression Methods Diagram

Identify the key steps and elements required for

transcription and processing of mRNA,
including but not limited to the role of the
promoter and splicing
...

Recognize the role of differential gene

transcription in development:
transcription factors and enhancers
tissue specificity of enhancers
Griffiths, 9th Ed., Ch. 8

Describe how selective nuclear RNA

processing can generate multiple isoforms by
alternate splicing
...

Compare and contrast each methods to assay

gene expression, including in situ hybridization,
reporter genes, quantitative RT-PCR, and RNASeq. Recall which methods yield spatial vs.
quantitative data.
...

Modern molecular genetics revolutionized the

study of vertebrate development
1982: Richard Palmiter and Ralph Brinster create
the first transgenic mouse.

Making a transgene
- artificial gene; must incorporate elements
critical for gene expression.
- A simple construction scheme has been
developed that provides the best transgene

Dev. Bio:

expression: each transgene contains a promoter,

an intron, a protein coding sequence,
transcriptional stop sequence and 3' polyA site.

Example transgene diagram

To make a transgene that expresses gene X

you must clone it into
a plasmid

What do you need to make a transgenic

mouse?
- Transgene
- Fertilized oocytes
- Pseudopregnant foster mother
- Method to screen for targeted embryos or pups

where are transgenes located when

implemented?
all cells of an embryo.

You are making a transgene, where do you

predict it will integrate into the genome?
A random chromosomal location

What can you learn from a transgenic mouse?

- Where are tissue/cell specific enhancers.
- When during embryonic time an enhancer is
functional.
- What does a specific cell lineage become.
- Where do cells of a specific lineage migrate.
Role of specific genes in development.

Where are tissue specific enhancers for a

specific gene?
in the enhancers for that particular tissue

Dev. Bio:

When/where is a gene expressed during

embryogenesis.

Why make a knock out mouse?

allows us to ask important questions about the
function of gene products.
learn what the role of a specific gene product is
by studying development in its absence
huge leap forward for mammalian developmental
biology

What do you need to make a knock-out?

- Embryonic stem cells
- Sequence and structure of the gene locus
- Targeting vector
- Method to screen for targeted ES cells
- Blastocysts
- Pseudopregnant foster mother
- Method to detect chimeric neonates

Pluripotent
stem cells; can become all of embryo, but not
extraembryonic tissues (eg. placenta,
membranes)

Embryonic stem cells (ESCs)

Pluripotent stem cells- can become all of embryo,
but not extraembryonic tissues (eg. placenta,
membranes)

Dev. Bio:

From the inner cell mass of the blastocyst

Can culture indefinitely in undifferentiated state
Discovery of viable culture conditions of mESCs
led to knock out/knock in and transgenic genetic
manipulations of mouse embryos
1998- James Thompson Univ. of Wisconsin
isolated and cultured human ESCs for the first
time

Homologous recombination
a type of genetic recombination also known as
crossing over that occurs during meiosis.
It also occurs in diploid cells under special
circumstances, this is important for ES cell
mediated knockouts.