Lab 3: Mystery Mutant Yeast & RD Mutants

How many ATP does glycolysis produce?

2 ATP

What is the optimum growth temperature of yeast?

30 degrees Celsius (can grow satisfactorily at room temp though)

How many ATP does the Krebs Cycle produce?

36 ATP

What were the small white colonies that grew?

A mutant of some kind, but they were capable of respiration because they grew on the YEPG plates

What do the red colonies represent?

Colonies that can respire

In petites, mtDNA is either completely ________ (P0) or consists of residual fragments of the ________ genome, usually reiterated. (P-)

Completely deleted grande

Should yeast be incubated in light or dark?

Dark

How does a respiratory deficient mutation occur?

Due to a variety of mutations affecting the genes and proteins of the mitochondria

What is the most common carrier of contamination?

Dust (carries bacteria and molds)

What is a fuel that is available to be used by RD mutants regardless of the availability of oxygen in the environment?

Glucose

Glucose is used as the energy source for glycolysis? In short, what is the pathway of ATP production and respiration?

Glucose --> Glycolysis --> pyruvate -->acetyl CoA --> Krebs cycle --> 38 ATP production

What are wild type yeast cells that respire normally known as?

Grandes they form large colonies

What is a conditional genetic disorder?

If an organism carries a harmful genetic mutation, it can still survive and sometimes even function normally if something in its environment can compensate for the genetic defect. in this condition, the organism may not appear to have a mutation at all. quick definition: the phenotype of the mutation is only observable under certain conditions

What can be done to lessen the effects of PKU mutation?

If identified at birth, then simple dietary changes can help overcome the negative effects. (all newborns are screened to help with this)

What is PKU?

In a normal person, the PKU gene product changes the amino acid phenylalanine into another chemical, People with PKU mutations, do not make that product and therefore cannot remove phenylalanine from the body. As a result, unwanted chemicals build up in the body. If untreated, can lead to severe mental retardation

What is mtDNA? Why is it so vulnerable?

It is mitochondrial DNA. It is round and single stranded like prokaryotic DNA. It is also called naked DNA. It has no histones or anything protecting it. nuclear DNA is not naked

If a yeast colony is able to grow in a glycerol environment, what is the conclusion you can make about the yeast colony?

It is not an RD mutant

Are the EtBr cells able to respire?

NO

Did the EtBr cells grow on the YEPG plates? What does that mean?

No, they are respiratory mutants

Is glycerol suitable for glycolysis?

No. But it can be converted and used in the Krebs cycle. SO, in our experiment if our yeast grow on YEPG they can respire, if they can't, then they can't respire.

On day 3, we took a sample of our growing mutant from YEAD plate and plated them on MV plates including different combinations of nucleotides. What happened vs what should have happened? What does it mean?

None of our plates grew at all. Should have had growth on plates containing the Adenine discs, because our mutant was an adenine mutant. It needed adenine to thrive.

How does cellular respiration normally occur?

Occurs through the Krebs cycle it is enzyme catalyzed - some of the enzymes are from nuclear DNA, most from mtDNA

What is a conditional mutation in humans?

PKU (phenylketonuria)

What are yeast cells that are unable to respire normally known as?

Petities or P+ small colonies - can only grow on fermentable carbon sources (glucose and fructose)

The frameshift mutations can cause what? When do they occur?

Respiratory deficiency inability to perform respiration occur when polymerase reads mRNA (changes the reading frame)

What is one physical trait that is commonly seen in respiratory deficient (RD) yeast mutants?

Small or Petite colonies

From the growing and non growing plates on the first day, we took sample and put them on YEAD and MV plates... What were the results? What do they mean?

So if it is on MV plate, our mutant is not going to grow ever If it is on a YEAD plate it will grow. Bakers yeast will grow on both plates

What is happening when a lawn of cells is formed?

The yeast are growing, each individual cell is growing into a colony, which is what is observable

When you streak cells and individual colonies are formed, how are these cells related to each other?

They are essentially dilutions of the same sample. There will be few cells in each streaked area than before.

Results of Day 1 (just dilutions & streaks on YEAD and MV)

YEAD: plates grew (this means that the YEAD plate has the nutrient that our mutant is deficient for) MV: did not grow (this means that our mutants are deficient for a nutrient unavailable on minimal media)

Do you need enzymes from nuclear and mitochondrial DNA for respiration?

YES, need both. If missing or mutation in one type, respiration is deficient.

What does YEPG stand for?

Yeast Extract Peptone Glycerol

What does YEPD stand for?

Yeast Extract Peptone (amino acid source) Dextrose (glucose is used as energy source for glycolysis)

Which is the most likely/ best hypothesis for the Mystery Mutant lab, if you see Yeast Strain X grow on YEAD but not on MV plates?

Yeast Strain X carries a conditional mutation that prevents it from growing on minimal media

Many colonies begin to grow, and since there are so many, they all overlap? Is this true?

Yes, colonies can grow very close to each other and overlap, making it difficult to count individual colonies. This is why we do serial dilutions

What type of progeny do P- give rise to?

a moderate proportion of petites

Our mystery strain X has a conditional mutation that prevents it from growing. What is this deficiency called?

a nutritional deficiency

What are frameshift mutations?

either addition or deletion of a nucleotide devastating to mtDNA

What does an intercalating agent do?

gets in between the bases in mtDNA and causes frameshift mutations EtBr is the one we used.

What type of progeny do HSp- give rise to?

nearly 100% petite descendants

In the Mystery Mutant lab, you will be controlling for the growing environment of yeast by adding certain combinations of ___________?

nucleic acids

What type of progeny do P0 give rise to?

only grande

When a mutation occurs, what happens to cellular respiration?

the Krebs cycle stops, so there are no more 36 ATP produced. The cell now has to rely on glycolysis which only produces 2 ATP.

What is the yeast mitochondrial genome known as?

the rho factor, it is represented by the Greek letter P

On the last day of the RD mutant lab, what was the purpose of plating the red colonies again?

to act as a control, we knew they should grow

What was the purpose of using diH2O on a disc?

use as a control