Project Report

2014 Submitted by:

Amna Jalil (17)

Submitted to: Dr.Nazia Jamil

[EFFECT OF UV RAYS &

PHOTOREACTIVATION ON THE COLONIAL MORPHOLOGY AND CATALASE ACTIVITY

OF BAKER’S AND WILD YEAST]

Contents

Abstract

Objectives

Introduction

Materials & Methods

Results

Discussion

References

Abstract

Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible

light, but longer than X-rays, that is, in the range between 400 nm and 10 nm. UV light causes

mutation in yeast cells and is lethal for its survival. Its main effect is on genes and DNA. In this

study two strains of yeast cells (one wild type isolated from soil and 2nd

one was baker’s yeast)

were taken and mutated with UV light for 1 minute to check the effect of UV on the colonial

morphology of yeast as well as on the catalase activity of yeast cells.Colonial morphology is

affected due to UV exposure and so does catalase activity which is surprisingly increased after

UV exposure. After photoreactivation, colonial morphology is same as it was before the

administration of UV and so does the catalase activity.

Objectives

To check the effect of UV on the colonial morphology of wild as well as baker’s yeast

To check the effect of Photoreactivation on the colonial morphology of wild as well as

baker’s yeast

To check the effect of UV on the catalase activity of wild as well as baker’s yeast

To check the effect of Photoreactivation on the catalase activity of wild as well as baker’s

yeast

Introduction

Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible

light, but longer than X-rays, that is, in the range between 400 nm and 10 nm. It is so-named

because the spectrum consists of electromagnetic waves with frequencies higher than those that

humans identify as the color violet. These frequencies are invisible to most humans except those

with aphakia. Near-UV is visible to a number of insects and birds.

Ultraviolet radiation in our environment is as common as sunlight. It generates genetic diversity

and kills cells.When a DNA molecule is damaged by radiation and the damage is not repaired

before the DNA replicates, the cell is likely to die. When a cell cannot divide to form viable

progeny, we say that it has suffered reproductive death. The cell may still be able to metabolize

and grow, but it cannot divide. If the radiation dose is high enough, cells can be killed outright --

metabolic death -- but other metabolic functions are far more resistant than reproduction.

In single-celled organisms such as yeast, other fungi, bacteria, and algae, mutations are an

important sublethal effect. Radiation also produces sublethal chromosomal changes and

stimulates genetic recombination.

Ultraviolet, this does not break the DNA chain outright, is selectively absorbed by the aromatic

rings of the purine and pyrimidine bases, so its energy, being more concentrated, is as damaging

as ionizing radiation. One particularly unpleasant result is the formation of pyrimidine dimers. In

this reaction, two adjacent pyrimidines in the same chain (T-T, C-C, or T-C) become covalently

bonded together. These dimers disrupt the local structure of the DNA double helix and prevent

normal DNA replication. They are not much better than a double-strand break, as far as the cell

is concerned.

Figure: Effect of UV on DNA

Photoreactivation (PR) is the recovery from biological damage caused by UV by simultaneous or

subsequent treatment with light of longer wavelength (PR light). Pyrimidine dimmers have been

shown to be induced in the deoxyribonucleic acid (DNA) of Saccharomyces cerevicae and other

yeasts by ultraviolet (UV) radiation. This damage can be repaired by a dark-repair mechanism

that alters or removes the dimmers Or by a light-repair system (photoreactivation) that

monomers them.

The splitting of the dimers is mediated by photoreactivating enzyme (PR), an enzyme that can be

removed in yeast by a mutation in the PHR1 gene.

Materials & Methods

Apparatus

Petri plates

Test tubes

Test tube stands

Incubator

Spreader

Micropipettes

Microtips

UV Illuminator

Aluminium foil

Spirit lamp

Wire loop

Flask

Reagent

3% H2O2

Media

YPD Agar

Sr No Ingredients Amount (g/liter) 1. Peptone 20

2. Dextrose 20

3. Yeast extract 10

4. Agar 15

For YPD Agar Peptone, Yeast extract and agar were dissolved in 700 ml of water and pH was set

at 6.5 and then autoclaved it. Dextrose was separately mixed with 300ml of distilled water and

after adjusting the pH autoclaved it for only 10 minutes and after cooling mixed with the rest of

the media.

YPD Broth

Sr No Ingredients Amount (g/liter) 1. Peptone 20

2. Dextrose 20

3. Yeast extract 10

For YPD Broth Peptone and Yeast extract were dissolved in 700 ml of water and pH was set at

6.5 and then autoclaved it. Dextrose was separately mixed with 300ml of distilled water and after

adjusting the pH autoclaved it for only 10 minutes and after cooling mixed with the rest of the

media.

Yeast Strains

Two yeast strains were used in this experiment.

1. One strain used was of baker’s yeast

2. One strain used was isolated from the soil sample taken from the garden of MMG

Procedure

Collection of the Yeast Sample

Soil sample was collected from the garden of the MMG department of the University of

the Punjab.

Baker’s yeast strain was purchased from the market

Spreading on YPD Agar plates

For soil sample, serial dilution was made and two dilutions (10-3

& 10-5

) were spread on

YPD Agar plates and incubated on 30°C for two days in order to achieve the crowding.

Figure: Crowding of yeast on 10-3 dilution of the soil sample

For Baker’s yeast sample, 0.5g of yeast was taken and mixed in about 10 ml autoclaved

YPD broth and incubated on 37°C for 2-3 hours for enrichment and after that spread on

the YPD agar plates and incubated.

Figure: Crowding of yeast from the baker’s sample

Isolation of Pure Strains

Yeast strains were identified by performing wet mount and then purified by performing streaking

on separate YPD agar plates.

Figure:Streaking of the pure sample from soil

Figure:Streaking of the pure sample of Baker’s yeast

Exposure of UV Radiations

YPD broth was poured in two test tubes and autoclaved. Then the single colony was picked from

both types of stains and was dissolved in different test tubes. After that, both test tubes were

exposed to UV rays in UV Illuminator for 1 minute and after the exposure were immediately

spread on YPD agar plates and covered with aluminum foil. After that, these plates were

incubated at 30°C for 48 hours.

Exposure of UV plus 2 min in sunlight

Photoreactivation requires exposure to a relatively intense source of visible light either in the sun

or artificial light. For photoreactivation, inoculated YPD plates after exposure to the UV

radiation, were exposed to the sunlight for 2 minutes and then incubated at 30°C for 48 hours.

Analyzing the Effect of UV & Photoreativation on Colony

Morphology

To check the effect of UV on colonial morphology, colonial morphology was noted

before and after the UV exposure.

To check the effect of photoreactivation on colonial morphology, colonial morphology

was noted after the exposure of UV irradiated plates to the intense visible light.

Analyzing the effect of UV & Photoreactivation on Catalase

Activity

To check the effect of catalase activity, catalase test was performed before and after the

UV exposure with the help of 3% H2O2 as a reagent.

To check the effect of catalase activity, catalase test was performed after the exposure of

UV irradiated plates to the intense visible light with the help of 3% H2O2 as a reagent.

Results

Effect of UV & Photoreactivation on the Colonial Morphology of

Baker’s Yeast Sample

Table: showing the effect of UV exposure & Photoreactivation on the colonial morphology of Baker’s yeast sample

Property Before UV Exposure After UV Exposure

(1 min)

After Photoreactivation

(2 min) 1. Form Circular Circular Circular

2. Elevation Convex Raised Convex

3. Margins Entire Entire Entire

4. Color Cream colored Cream colored Cream colored

5. Surface Smooth & Shiny Rough & Matt Smooth & Shiny

Figure: Colony morphology of Baker’s yeast before UV exposure

Figure: Colony morphology of Baker’s yeast after UV exposure

Figure: Colony morphology of Baker’s yeast after photoreactivation

Effect of UV & Photoreactivation on the Colonial Morphology of

Wild type Yeast Sample

Table showing the effect of UV exposure & Photoreactivation on the colonial morphology of wild type yeast sample

Property Before UV Exposure

After UV Exposure (1 min)

After Photoreactivation

1. Form Circular Circular Circular

2. Elevation Convex Raised Convex

3. Margins Entire Entire Entire

4. Color Cream colored Cream colored Cream colored

5. Surface Smooth & Shiny Rough & Matt Smooth & Shiny

Figure: Colony morphology of wild yeast before UV exposure

Figure: Colony morphology of wild yeast after UV exposure

Figure: Colony morphology of wild yeast after photoreactivation

Effect of UV on the Catalase Activity of Baker’s Yeast Sample

Figure: Catalase activity of Baker’s yeast before UV exposure (Positive)

Figure: Catalase activity of Baker’s yeast after UV exposure (increased)

Figure: Catalase activity of Baker’s yeast after photoreactivation

Effect of UV on the Catalase Activity of Wild Yeast Sample

Figure: Catalase activity of wild yeast before UV exposure (Positive)

Figure: Catalase activity of wild yeast after UV exposure (increased)

Figure: Catalase activity of wild yeast after photoreactivation

Discussion

It is well known that Ultraviolet radiations are lethal for yeast cells. UV radiations cause

mutations in yeast cells. A certain amount of mutational changes in the genome occurs as a

natural process, though the probability is low. These radiations affect the cell growth rate as well

as survival rate. These radiations also affect the colonial and cellular morphology of yeast cells.

UV radiations also affect the enzymatic activity of yeast cells.

After the UV exposure of 1 minute, In case of colonial morphology of baker’s yeast the form

remains same i.e., circular, elevation is changed from convex to raised. Margins of colonies are

not much affected and remain entire. Color of colonies is also not changed after UV exposure.

Surface of colonies is affected and changed from smooth & shiny to matt and rough. Size of the

colonies is not much changed after the treatment with UV.

After the UV exposure of 1 minute, the results of the colonial morphology of the wild yeast

strain were similar to those with the baker’s yeast strain.

In both strains of yeast survival rate also decreases and the effect of UV is mainly on the genetic

level i.e., on genes.

For the analysis of enzymatic activity of catalase, catalase test was performed both before and

after the UV exposure and results were noted. Both yeast strains were catalase positive and after

the exposure of UV, their catalase activity increases. The effect of UV irradiation on the catalase

activity of an aqueous yeast suspension was divisible into 4 periods. First, the period during

which the cells lost their ability to form colonies, but during which no change in catalase activity

was noted. Second, the period during which a considerable rise in catalase activity occurred.

Third, a rather long period during which irradiation led to no diminution in the catalase activity

of the maximally active suspension. Fourth, the period of photoinactivation of the intracellular

enzyme, which was quite similar to that of the crystalline enzyme in vitro. So, by this analysis,

both samples were in second period.

As long as photoreactivation is concerned, colony morphology was same as it was before the

exposure of UV and so does the catalase activity of both the strains of yeasts.

References

Aldous Jg, Stewart Dkr. The effect of ultraviolet radiation upon enzymatic activity and viability of the

yeast cell.Can J Med Sci. 1952 Dec;30(6):561–570.

E. Tomkinson, S. Wei, Z.Y. You. Nucleotide excision repair in yeast: recent progress and implications.

1998. Nucleic Acids Mol. Biol., 12:125-139.

Kaplan Jg.The alteration of intracellular enzymes. II. The relation between the surface and the biological

activities of altering agents. J Gen Physiol. 1954 Nov 20;38(2):197–211.