Exam 1

The exam will be all multiple choice.

Youll be using a scantron form and will need a No. 2
Pencil.
All academic questions will be derived from the power
points shown in class and available on OnCourse.
Two calculations will be covered on the exam: Serial
Dilutions and Conjugation Mapping in prokaryotes.
Exam one will not test over this power point.

Respiratory Mutants
Cellular Respiration is the most prevalent and efficient
catabolic pathway for the production of ATP, in which
oxygen is consumed as a reactant along with the organic
fuel.
Organic Compound + O2 CO2+ Water + Energy

Respiratory Mutants
Many carbon/energy sources can be used to fuel
respiration including: Carbohydrates, Fats, and Proteins.
It is often useful to follow Glucose through the process.
C6H12O6 + 6 02 6 CO2 + 6 H2O + Energy
The energy can be in the form of ATP or heat.

Respiratory Mutants
Oxidation and Reduction of molecules plays an important
role in respiration.
Redox reactions involve the transfer of electrons from one
reactant to another.
One reactant will lose electrons and that reactant will be
oxidized.
The other reactant will gain electrons and it will be
reduced.

Respiratory Mutants
Adding electrons to a reactant is called reduction
because the addition of electrons to a molecule will
reduce the positive charge of that reactant.
The electron donor is called the reducing agent because
it reduces the reactant accepting the electrons.
The electron acceptor is called the oxidizing agent as it
oxidizes the other reactant by taking electrons from it.

Respiratory Mutants

A mnemonic device that works here is:

Leo was a German.
Leo = lose electrons is oxidation
Ger = gain electrons is reduction
Glucose becomes oxidized and O2 becomes reduced.

Overview of ATP Production

Overview of Glycolysis

Glycolysis
The only important players in the above schematic are:
Hexokinase: an enzyme whose substrates are Glucose
and ATP and starts the Glucose catabolism process by
phosphorylating Glucose.
Glyceraldehyde 3 phosphate: a 3 carbon molecule
that will produce 1 ATP and 1 NADH2 in glycolysis.
Pyruvate: converted into Acetyl CoA which can feed
directly into Krebs cycle allowing the turning of the Krebs
cycle or Respiration.

Respiration occurs in the Mitochondria.

Overview of Respiration

Overview of Respiration
Acetyl CoA feeds Krebs
cycle allowing it to turn.
The output of a single
cycle of Krebs is 1
molecule of FADH2, 3
molecules NADH2, and 1
molecule of ATP through
substrate
phosphorylation.

Overview of Respiration
The reducing power of
FADH2 and NADH2 can
be converted into ATP
through oxidative
phosphorylation.
A 6 carbon Glucose
molecule yields 2 three
carbon pyruvate
molecules during
glycolysis.

Overview of Respiration
For every glucose
molecule that enters into
glycolysis, the krebs
cycle will turn twice.

ATP Production

ATP Production
Per glucose molecule, we see the following ATP
production assuming all reducing power is converted to
ATP.
Glycolysis: 2 ATP
Respiration (Krebs cycle): 36 ATP
Which process provides more energy for a growing cell?
Obviously respiration is the preferred way to glean energy
from carbohydrates such as glucose.

Mitochondrial DNA
In 1952, Boris Ephrussi observed both small and large
yeast colonies growing on agar media.
He coined the phrase petite to describe the small
colonies.
The larger colonies are then termed to be grandes.
The petite trait was shown to be inheritable from parent to
offspring during vegetative reproduction or during a cross
between 2 petite parents. The trait was not passed on to
offspring during a cross between grandes and petites.

Mitochondrial DNA
Further experimentation with petites revealed that some
petites, when mated to a grande, would produce 2 petite
offspring and 2 grande offspring.
These petites followed Mendels theory of independent
assortment and were called segregational petites as they
segregated in a Mendelian fashion.
We now have two types of petites. Those following
Mendelian assortment and those that dont.
How can we explain these two petites?

Mitochondrial DNA
Remember that a colony observed on a petri dish is the
result of one cell being deposited on the agar and then
allowed to grow into many cells producing the colony.
If one colony is smaller than another, what would you say
about the rate of growth of those two colonies?
Would an organisms rate of growth be affected by its
ability to respire?
Glycolysis yields 2 ATP and Respiration yields 36 ATP.

Review of Yeast Life Cycle

Yeast live happily as either 1N or 2N organisms.
As 1N organisms, they have either an a or mating
type.
An a can mate to an creating a 2N cell.
The 2N cell can undergo meiosis and produce four 1N
offspring.
2 of the offspring will be the a mating type and 2 will be
the mating type following Mendels theory.

Review of Yeast Life Cycle

Observed Mating Results

PxG

Observed Mating Results

PxG

Notice the mating on the left produces 2 petite offspring

and 2 grande offspring. These follow independent
assortment.
The mating on the right, however, produced only grande
offspring. If we mate the grandes back to a petite to
perform a test cross, we still see only grandes.
This is a bit of a conundrum that needs to be explained.

Mitochondrial Genomes
It was previously known that mitochondria were the sites
of respiration.
What wasnt known at that time was that mitochondria
have their own DNA genome.
If fact, many scientists today would argue that
mitochondria and chloroplasts have evolutionary roots as
prokaryotic organisms.

Mitochondrial Genomes
The theory is that at some point in time one prokaryotic
cell engulfed another.
Rather that digesting that cell, the cell continued to live in
the cytoplasm of the host cell and became a membrane
bound organelle.
What evidence supports such a claim?
Lets review the characteristics of prokaryotic cells and
compare them to mitochondria or chloroplasts.

Mitochondrial Genomes
Prokaryotic cells have a small circular genome lacking
histones, etc.
Prokaryotic cells translate their mRNA on 70s ribosomes.
Prokaryotic cells have no membrane bound organelles.
Mitochondria have a small circular genome lacking
histones, etc.
Mitochondria translate their mRNA on 70s ribosomes.
Mitochondria have no membrane bound organelles.

Mitochondrial Genomes
We now see that mitochondria function with genetic input
from two different sources.
Some proteins required for mitochondrial function are
encoded in the nucleus of the cell. These proteins are
targeted to and transported to the mitochondria.
Other proteins required for mitochondrial function are
coded on the prokaryotic like genome of the
mitochondria.
Can these differences explain our P x G conundrum?

Analyzing P x G
We know that functioning mitochondria are required for
respiration.
We know that there are 2 genetic sources for proteins
required for functional mitochondria. One nuclear source
and one mitochondrial source.
Nuclear genes are transmitted to offspring through
meiosis.
Mitochondrial genes are transmitted to offspring through
simple binary fission.

Analyzing P x G
Of these two methods of gene transmission, which one
would most likely follow Mendels law of independent
assortment? The nuclear genes transmitted via meiosis.
Perhaps the difference between the transmission of these
two genomes can explain our P x G conundrum.
Nuclear mutations that can knock out mitochondrial
functions would easily explain the results we see yielding
two petite offspring and two grande offspring.
The other cross is not so clear.

Analyzing P x G
How can we explain the fact that some petites, when
crossed to grandes, simply lose the petite trait and that
trait cant be recovered even after performing a test cross
back to a petite.
When 1N yeast cells mate, they fuse together to become
one 2N cell.
The cytoplasms of the two cells mix including the
mitochondria found in each cytoplasm.

Analyzing P x G
We mated a petite with non-functional mitochondria due
to mutations in the mitochondrial genome.
The nuclear genome is wild type.
During mating of a and mating types, cells fuse and
mix their cytoplasms distributing the functioning and nonfunctioning mitochondria evenly throughout.
During meiosis, the 2N cell divides twice splitting its
genome and cytoplasms.

Analyzing P x G
All progeny cells get at least one functioning mitochondria
Mitochondria can proliferate using autonomous
replication
All progeny cells are now grandes and respiratory
capable.
Mating these cells back to a petite as a test cross will
yield the same result, all progeny who are grandes.
These types of petites are called cytoplasmic petites
because the mitochondria, or mitochondrial DNA, reside
in the cytoplasm, not the nucleus.

Analyzing P x G

Creating Respiratory Mutants

We will use Ethidium Bromide to mutate the mitochondrial
genome in Saccharomyces, a eukaryotic yeast cell.
Ethidium Bromide belongs to a class of mutagens called
the Intercalating Agents.
These agents are planar molecules that mimic base pairs
and are able to slip themselves (intercalate) in between
the stacked nitrogen bases of the double helix.

Creating Respiratory Mutants

Ethidium bromide intercalation requires the DNA helix to
relax or unwind to allow insertion of the planar molecule.
Therefore, Ethidium bromide intercalates into the naked
DNA of the mitochondria more readily that the more
tightly coiled nuclear DNA. Nuclear DNA has clothing in
the form of Histones and other proteins that help in
condensing the DNA molecules and keeping them tightly
coiled.

Creating Respiratory Mutants

Ethidium bromide can cause a DNA polymerase to
errantly insert or delete a base in the DNA sequence.
This creates a Frameshift mutation.
What affect does a Frameshift mutation in a gene have
on the resulting protein product?
How different will the amino acid sequence of the
resulting protein be compared to the wild type sequence?

Creating Respiratory Mutants

Creating Respiratory Mutants

Frameshift mutations have a very dramatic affect upon
the folding of the protein product.
Frameshift mutation will usually cause the resulting
protein to fold improperly and that protein will lose
whatever function it was intended to have.
Frameshift mutations in mitochondrial DNA will leave the
mitochondria non-functional creating a respiratory mutant.

Intercalated Ethidium Bromide

Respiration Assay
There are many reasons why a yeast colony would be
smaller than others seen on the petri dish.
Suppose a mutation occurred in the tryptophan synthesis
enzymes that caused the synthesis of tryptophan to be
only 0.1% efficient.
Would that colony grow slowly because it is constantly
waiting the synthesis of a tryptophan molecule? Yes.

Respiration Assay
Remember, a petite colony grows slowly because it cant
respire and therefore it loses out on the 36 ATP produced
during the Krebs cycle.
It can grow utilizing the 2 ATP generated in Glycolysis by
the catabolism of Glucose.
It simply has a slow growth rate because it has to wait for
ATP to be formed and appears as a petite on media.
We can use Glycerol as an alternative Carbon/Energy
sources to test the organisms ability to respire.

Glycerol Assay
Why cant petites grow on a Glycerol agar plate?
Petites survive utilizing glycolysis alone.
The first enzyme of glycolysis is Hexokinase which
phosphorylates glucose to start the process.
Is Glycerol a suitable substrate for Hexokinase? No, it is
a different molecule entirely.
Therefore, you take away the only source of ATP
production the petite could utilize = no growth.

Glycerol Assay
How do respiratory capable yeast survive without glucose
(and glycolysis) and thrive on a glycerol C/E source?
Remember that suitable molecules for respiration where
carbohydrates, fats, and proteins.
Glycerol can fuel Krebs cycle and allow the production of
a generous amount of ATP.
Glycerol can shunt into Glycolysis, produce pyruvate,
acetyl CoA, and enter Krebs cycle.

Glycerol Assay

Glycerol Assay
The important features of this pathway are:
Glycerol Kinase adds a phosphate to the 3 Carbon
Glycerol.
The enzyme Glycerol phosphate dehydrogenase
consumes NAD+ while making dihydroxyacetone
phosphate.
The end product of this reaction is Glyceraldehyde 3
Phosphate which is an intermediate of Glycolysis.

Overview of Glycolysis

Glycerol Assay
The enzyme Glycerol phosphate dehydrogenase
consumes NAD+ while making dihydroxyacetone
phosphate. A further NAD+ is consumed in glycolysis after
glycerol has entered that pathway (2 total).
Glycolysis of Glucose consumes only 1 NAD +.
Petite cells attempting to utilizing Glycerol as a C/E
source soon run out of NAD+ because it is not being
regenerated by ETS since respiration isnt occurring in
petites.

Glycerol Assay: The Bottom Line

Petites can not grow on a glycerol carbon energy source.
Grandes can utilize glycerol as a C/E source and will
grow using the energy gleaned from Krebs cycle.