Bio 211 lab 13&14 PDF

Preview

Summary

Lab 13& 14...

Description

April 9, 2019

Complementation of Yeast Strains Genetically Crossed Abstract In this experiment, 4 different yeast strains (HA1, HA2, HB1, and HB2) were crossed with each other. Over the period of one week, the diploid yeast colonies grew and developed, and then the phenotypes were observed. The purpose of this experiment was to identify how the mutated yeast strains will affect the phenotypes, specifically growth and color, of the reproduced diploid yeast colonies. We observed the mutated yeast strains based on the hypothesis that the mutations are caused due to the modification of a protein sequence. results showed that HA2 + HB2 and HA1 + HB1 had a pink phenotype, also having the 2 scores with the largest amount of growth. The strains HA1 + HB2 and HA2 + HB1 both had a cream phenotype, also having the 2 scores with the lowest amount of growth. Introduction Yeast are unicellular fungi, meaning that in disregard to reproduction, there is only one cell in each single yeast. Yeast cells can reproduce both asexually and sexually. They reproduce asexually through a process called budding, that allows them to cluster into colonies of many yeast cells. They continue reproducing asexually, until the opposite mating type signals them to transform into gametes, or shmoos, that complement the opposite mating type. Once this happens, the shmoos fuse together and form a zygote. It was hypothesized that mutations in alleles of the genes are caused due to the modification of proteins by changing the sequence, therefore, changing the protein structure and function. It was predicted HA1 x HB1 would produce a pink color and would grow slow, HA1 x HB2 would produce a cream color and grow fast, HA2 x HB1 would produce a cream phenotype

and grow fast, and HA2 x HB2 would produce a pink color and grow slow. This is seen by the table 12-1 below. Table 12-1. Genotypes and phenotypes of haploid and diploid yeast strains. Haploid Diploid Yeast Alleles of the Alleles of the Color (Pink Yeast Strains Strains ADE 1 Gene ADE 2 Gene or Cream)

HA1, HB1 HA2, HB2 --

----

--HA1 X HB1

HA1 X HB2 HA2 X HB1 HA2 X HB2

Growth on MV (slow or fast)

ade 1 ADE 1 ade 1 ade 1

ADE 2 ade 2 ADE 2 ADE

Pink Pink Pink

Slow Slow Slow

ade 1 ADE 1 ADE 1 ade 1 ADE 1 ADE

2 ADE 2 ade 2 ade 2 ADE 2 ade 2 ade 2

Cream Cream Pink

Fast Fast Slow

1

Methods This experiment took a total of two weeks long, in order to observe the growth of the yeast strain’s. Each strain of yeast was placed on a YED, a dish with an adenine nutritional supplement, and labeled. To cross and mate these strains, a sterile toothpick was used to transfer the first strain in the cross to a separate part of the YED, and another toothpick was used to transfer the second strain on top of the first strain. The strains were mixed together in the petri dish with the sterile toothpick. Once the four strains had been crossed, the diploid yeast strains were transferred to a MV plate, using sterile toothpicks, and each diploid strain was labeled. Both the YED and MV plates were wrapped in Parafilm to be sealed, and then incubated. The night before the 2nd part of the experiment, where they were placed in a refrigerator to stop growth. The plates were then taken out and the phenotypes of the diploid yeast strains were observed, by color and growth. The yeast cells were then placed under a microscope where the stages of the yeast’s asexual and sexual life cycles were observed. The independent variable in

this experiment was the crossing combinations of the yeast (HA1 + HB1, HA1 + HB2, HA2 + HB1, HA2 + HB2). The dependent variable was the phenotype of the diploid strains. Results Table 13-1. Observed results of the phenotypes of diploid yeast colonies. Cross HA1 + HB1 HA1 + HB2

HA2 + HB1

HA2 + HB2

Color and Notes Strong pink color, no cream colonies present. Cream colonies, with splatter of pink in the middle colonies. Cream colonies with a small amount of light pink in the middle. Dark Pink colonies with no cream colonies observed.

Growth 3 2

1

4

As you can see, the results shown in Table 13-1, HA2 + HB2 and HA1 + HB1 had a pink phenotype, they had the 2 scores with the smallest amount of growth. The strains HA1 + HB2 and HA2 + HB1 both had a cream phenotype, also having the 2 scores with the largest amount of growth. There was an anomaly, a reversion occurred, meaning that the yeast were recovering from the mutation. The two crosses HA1 + HB2 and HA2 + HB1 were creamy with a small amount of pink meaning that they were recovering.

Discussion The hypothesis states that, mutations in alleles of the genes are caused due to the modification of proteins by changing the sequence, therefore changing the protein structure and function. The hypothesis was supported by the results exhibited in Table 13-1. Due to the protein sequence being altered, the mutated diploid colonies would show little growth, and appear pink due to the build-up of the reactants (CAIR or AIR). It was predicted that this pattern would be seen in the

phenotypes of the cross between the mutated strains HA1 + HB1 and HA2 + HB2. This same pattern is seen in Table 13-1, where HA1 + HB1 and HA2 + HB2 both appeared pink and showed very little growth. It was predicted that the non-mutated diploid strains, HA1 + HB2 and HA2 + HB1, would appear with a normal phenotype of yeast, which is cream, and would show fast growth because they are able to synthesize AMP and do not accumulate CAIR or AIR. This is also seen in Table 13-1, where HA1 + HB2 and HA2 + HB1 appeared cream and showed fast growth. The hypothesis was supported, as seen by the specific mutation in the yeast. There was an anomaly, a revision occurred in the cream colored yeast cross. In the cream color there was a liitle dot of pink, meaning that the yeast were recovering. Perhaps this occurred because when we crossed the yeast we did not put the same amount of each, causing a reversion to occur, or perhaps we didn’t keep the our plate of YED agar properly closed. From this experiment, it can be concluded that when the amino acid structure is changed in the primary structure of a protein, the protein is also changed. Therefore, giving the protein a different function that causes a mutation in the alleles of a gene coded for that protein. In humans, these mutations are what lead to different genetic disorders, like Alkaptonuria, PKU, cystic fibrosis, sickle cell anemia, etc.

DNA Analysis 1. After observing the electrophoresis results, Suspect 1’s DNA ( the middle line) lined up and matched exactly with the DNA found from the crime scene, whereas Suspect 2’s

DNA ( the top line that is very dull) did not line up with the DNA found at the crime scene. 2. It is easier to establish innocence than guilt. This means that its easier to establish a mismatch rather than a match because it is easier to identify a line that does not match the DNA at the crime scene, due to its position, rather than DNA that does, because there could be a chance that the suspect has the same DNA profile as someone else. Or perhaps that there is another explanation as to why that person’s DNA is at the crime scene. Fig 15.2- picture of the DNA bands on the gel

References

Marion, A.L, L.L Haas, R.W Preszler. 2018. Cellular and Organismal Biology Individuals lab Manual, 12th edition, Macmillan Learning Curriculum Solutions, MI....