TA: Lauren Javier
FALL 2014
Discussion D10, D11, D12
Lectures 23-25 Study Guide
I have organized some terms and topics that I think are important. This does not mean that other topics
mentioned during lecture or in the book will not be tested. This guide is meant to clarify and emphasize certain
points, NOT to list everything you need to know. I will focus on tying things together across lectures, and giving
real-life examples of the biological principles that we are learning. Details that I include that I think will be
helpful, but that you don’t need to know, I will write in green. Questions to think about I will write in blue.
Lecture 23: Regulation of gene expression
Keep in mind that all the cells in your body contain the same DNA. How is it, then, that your skin cells are so
different from your muscle cells? Because the genes are regulated differently in each cell type!!
DNA
1. Chromatin modifications:
a. Acetylation of histone tails makes chromatin more flexible, allowing transcription factors better
access to DNA, thereby facilitating transcription
b. Methylation of DNA impedes transcription factors from reaching DNA, thereby reducing
transcription
One important example of DNA methylation is shown in Prader-Willi Syndrome and Angelman
Syndrome. In both of these diseases, one allele was silenced by genomic imprinting via
methylation.
2. Transcription factors:
a. In eukaryotes, particular proteins are needed in order for RNA polymerase to bind the
promoter. Without these proteins, transcription will not occur. Therefore, changing the amounts
of these proteins will have a direct impact on transcription of a gene.
b. Activators: bind to enhancer regions to facilitate transcription
i. Different genes have different combinations of control elements within their enhancers
ii. Different cell types have different activators present
RNA
1. Alternative splicing: different combinations of exons are selected from the primary RNA transcript to be
translated. This allows a single gene to code for many different proteins.
2. mRNA degradation
a. mRNA doesn’t last long in the cytoplasm, despite the 5’ cap and poly-A tail
b. The cap and tail are soon removed, allowing the nucleases present in the cytoplasm to digest
the rest of the mRNA until there is nothing left
c. Different genes may have different lengths of 3’ tail, altering the rate of degradation
3. Translation initiation: can be blocked, preventing attachment of mRNA to ribosome, and thus
preventing translation from occurring
Protein
1. Glycosylation: addition of sugars destined for the plasma membrane (remember this from earlier in the
quarter? where does this occur?)
2. Cleavage, folding, etc.
3. Addition or removal of phosphate groups to activate or deactivate a protein
4. Transportation to final location (e.g. plasma membrane)
5. Degradation: ubiquitin / proteasome system
Lecture 23: modified from materials prepared by Carley Karsten, 2013
1
TA: Lauren Javier
FALL 2014
Discussion D10, D11, D12
a. A protein is marked for destruction by attaching a ubiquitin “tag” to it
b. Ubiquitin is recognized by proteasomes
c. Proteasomes degrade the tagged protein
Lecture 24: Introduction to DNA Recombinant Technology
Biotechnology: The manipulation of organisms or their components to make useful products
1. Relies on recombinant DNA, a nucleotide sequence from two different sources, often two different
species, combined to in vitro (in a test tube) into the same DNA molecule. This has given rise to the
development of powerful techniques for analyzing genes and gene expression!
Clone
1. Definitions:
a. A lineage of genetically identical individuals or cells
b. A single individual organism that is genetically identical to another individual
c. To make one or more genetic replicas of an individual cell
d. To produce multiple copies of a gene. Why would we want to do this?
2. Cloning a gene
a. Bacterial cells contain plasmids, which are small, circular DNA molecules that replicate
separately from the bacterial chromosome. These contain only a small number of genes that
are not necessary for life. To clone pieces of DNA, DNA from another source (contains a gene or
genes of interest) is inserted into the plasmid (cloning vector)= Recombinant DNA
i. Restriction enzymes cut DNA molecules at specific DNA sequences called restriction
sites. Restriction enzymes can make many cuts (as long as it finds that particular
sequence) and make many cuts yielding restriction fragments). Restriction enzymes cut
DNA in a reproducible way where all copies of a particular DNA molecule will always
have the same set of restriction fragments when exposed to the same restriction
enzyme.
ii. Restriction enzymes were developed as a defense mechanism against phages. They’d
recognize a foreign DNA and remove it so that the virus doesn’t take over the cell. How
would a bacterial cell protect itself from their own restriction enzymes?
iii. Most restriction sites are palindromic (same in both directions, 5’  3’). Restriction
enzymes make staggered cuts (usually done this way) that produce fragments with
“sticky ends” that bond with complementary sticky ends of other fragments. The
plasmid and DNA of interest is mixed together and the sticky ends combine. Through
hydrogen bonds. The bonds between the plasmid and DNA are sealed using DNA ligase.
b. The plasmid (recombinant DNA) is then inserted into a bacterial cell producing a recombinant
bacterium. This cell reproduces through repeated cell divisions to for a clone of cell that include
the plasmid!
Amplifying DNA using the Polymerase Chain Reaction (PCR)
1. PCR can produce many copies of a specific DNA fragment! We could use this tool to study a specific
gene/DNA fragment even though the actual amount of DNA is very small. This is great when studying
ancient (and extinct) animals, forensic science, viruses, etc.
2. It requires: template DNA, dNTPs, water, DNA polymerase, and primers
3. Three step cycle
a. Heating: The reaction mixture is heated to denature (separate) the DNA strands
b. Cooling: The DNA strands are cooled to allow annealing (hydrogen bonding) of short, single
Lecture 23: modified from materials prepared by Carley Karsten, 2013
2
TA: Lauren Javier
FALL 2014
Discussion D10, D11, D12
stranded DNA primers complementary to sequences on opposite strands at each end of the
target sequence.
c. Replication: A heat –stable DNA polymerase extends the primers in the 5’  3’ direction. What
would happen if a standard DNA polymerase were used?
Gel Electrophoresis is used to visualize DNA molecules.
1. This gel acts as a molecular sieve to separate DNA molecules based on their size, electrical charge, and
other physical properties.
a. Because nucleic acid molecules are negatively charged, they toward the positive pol. As the
DNA molecules move, the agarose (gel) impedes larger molecules than it does shorter ones.
This way, smaller molecules move faster and farther than larger molecules and molecules then,
are separated by size.
b. Who’s your daddy? In paternity tests, restriction enzymes are used to cut the DNA up into
fragments, run it through PCR, and then use gel electrophoresis. Remember, for any particular
DNA it will have the same restriction fragments. When comparing between the child and
potential fathers, researchers can compare the bands that represent the restriction fragments.
The father will have bands that match the child.
Cloning Mammals: Dolly
1. Dolly was the first mammal to be cloned and this was done through nuclear transplantation: the
nucleus of an unfertilized or fertilized egg is removed and replaced with a nucleus of a differentiated
(i.e., mammary) cell.
2. The genetic makeup of Dolly was identical to the animal supplying the nucleus (Sheep #1) but differed
from that of the egg donor (Sheep #2) and the surrogate mother (Sheep #3). Where did her
mitochondrial DNA come from?
3. Researchers suspected that there was something wrong in the way she was aging. She developed
problems that are normally seen in older sheep. They found that her telomeres were unusually short.
What are some other problems associated with animal cloning?
Stem Cell: relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized
cells of one or more types.
1. Embryonic stem (ES) cells: cells isolated from early embryos at the blastocyst stage. They can
differentiate into all cell types
2. Adult stem cells: Cells that generate some cell types, replacing non-reproducing specialized cells.
3. Induced pluripotent stem (iPS) cells: Treated differentiated cells that are reprogramed to act like ES
cells.
Lecture 25: Genetic Basis of Development
Development
1. The transformation from a zygote into an organism (Remember emergent properties from our very first
lecture? Cells  tissues  organs  organ systems  whole organism) is orchestrated by gene
expression and results from 3 interrelated processes:
a. Cell Division
b. Cell Differentiation: The process in which cells become specialized in structure and function.
Two sources of information “tell” a cell which genes to express at any given time during
embryonic development.
i. Cytoplasmic determinants: The cytoplasm of an unfertilized egg is not homogenous.
mRNAs, proteins, other substances, and organelles are distributed unevenly and this
Lecture 23: modified from materials prepared by Carley Karsten, 2013
3
TA: Lauren Javier
FALL 2014
Discussion D10, D11, D12
unevenness influences the developing embryo. The combination of cytoplasmic
determinants in a cell helps determine its developmental fate by regulating the cell’s
gene expression during differentiation. Cytoplasmic determinants include transcription
factors.
ii. Environment around the cell: Signals from neighboring cells, binding of growth factors,
etc. cause changes in target cells (induction). In induction, signal molecules from
embryonic cells cause transcriptional changes in nearby target cells.
c. Morphogensis: The physical process that gives an organism its shape.
2. Determination: The point to which an embryonic cell is irreversibly committed to becoming a particular
cell type. This step precedes differentiation!
a. Master regulatory genes produce proteins that commit a cell to a particular cell type.
i. Ex: MyoD protein (gene is referred to as myoD; Note the capital “M” in the protein vs.
the lowercase “m” in the gene. This is to differentiate between the two) is a
transcription factor that binds to enhancers of various target genes. This protein
stimulates the myoD gene further and activates other genes encoding other musclespecific transcription factors. This in turn activates genes for muscle proteins.
3. Pattern Formation: The development of a spatial organization of tissues and organs.
a. Maternal effect genes: Encodes for cytoplasmic determinants that initially establish the axis of
the body of the Drosophila. If this gene has a mutation in the mother, it results in a mutant
phenotype in the offspring, regardless of the offspring’s own genotype. Because maternal
effect genes control the orientation (or polarity) of the egg, they’re also called egg-polarity
genes.
b. Morphogens: Substances that establish axis formation or other features in a gradient.
i. Morphogen gradient hypothesis: gradients can establish the embryo’s axes.
1. Head: very high bicoid concentration
2. Thorax: medium concentration
3. Abdomen: low concentration
ii. Bicoid: morphogen that determines the head structures of an organism. In a nonfunctional bicoid, the embryo lacks the front half of its body and has posterior structures
at both ends.
Embryonic Development
1. Fertilization: The fusion of sperm and egg, which forms a zygote
2. Cleavage: Series of cell divisions divide, or cleave, the zygote into a many-celled embryo. These
cleavage divisions, which typically are rapid and lack accompanying cell growth, convert the embryo to
a hollow ball of cells called a blastula
a. The cell cycle consists primarily of the S (DNA synthesis) and M (mitosis) phases. Cells
essentially skip the G1 and G2 phases and little or not protein synthesis occurs.
b. Cleavage partitions the cytoplasm of one large cell into many smaller cells called blastomeres.
The blastula is a ball of cells with a fluid-filled cavity called a blastocoel.
c. The presence of yolk influence the pattern of cleavage
i. Vegetal pole: more yolk; slower cell division
ii. Animal pole: less yolk
d. Why do we have cleavage or this period of rapid cell division?
3. Morphogenesis: The process by which cells occupy their appropriate locations
a. Gastrulation: The blastula folds in on itself, rearranging into a three-layered embryo, the
Lecture 23: modified from materials prepared by Carley Karsten, 2013
4
TA: Lauren Javier
FALL 2014
Discussion D10, D11, D12
gastrula.
i. Ectoderm: forms the outer layer
ii. Endoderm: lines the embryonic digestive compartment or tract
iii. Mesoderm (in animals with bilateral symmetry): between the ectoderm and endoderm
b. Organogenesis: Local changes in a cell shape and large-scale changes in cell location generate
the rudimentary organs from which adult structures grow.
i. The notochord forms from the mesoderm. This gives rise to the spinal cord. The neural
plate gives rise to the brain and nerves.
Lecture 23: modified from materials prepared by Carley Karsten, 2013
5