Genotoxicity of Goji Berry (Lyciumbarbarum) In Vivo Mammalian Cells
SHY1
1. Mutations in the SHY1 deletion in
Saccharomyces cerevisiae and Effects on
Cellular Respiration
NabeelRashid, Tauseef Ahmed, Abdul R. Khan
Department of Biology, New York Institute of Technology, Old Westbury, NY
_____________________________________________________________________________________
Dr. Gavin McStay
Biomedical Research II
May 12th
, 2016
2. 1
Abstract
Yeast (Saccharomyces cerevisiae) are model organism for the study of eukaryotic cells for
various reasons. Yeast are eukaryotic organisms that fall under the Fungus Kingdom. The key
reason they are used by scientist is due to their rapid rate of division, about every 90 minutes.
These yeast also are easy to work with due to them being small in size and inexpensive. Their
lack of complex organs and body system make it easier to work with than other mammalian
cells. SHY1 in yeast cells is required for efficient cytochrome c oxidase biogeneis in the
mitochondrial inner membrane. SHY1 also plays a role in the insertion step for Haem a. The
purpose of this experiment was to see if yeast cells would grow in the background of a mutated
SHY 1 gene. Results showed that absorbance was high in the mutated state and growth was
positive. Furthermore, results indicated that yeast were able to undergo aerobic respiration with
the mutated SHY 1 gene. This is indicative of the fact that the results of a mutation in yeast may
not be the same for their human homolog counterparts. This is because SURF1 mutations in
humans are linked to Leigh syndrome and are more disastrous than SHY1 mutations.
Introduction
Cytochrome c oxidase (complex IV) is an enzyme encoded by both nuclear and
mitochondrial DNA. Defects in the assembly process of complex IV can lead to severe disorders
in humans such as Leigh syndrome (Cytochorome). SHY1 is an assembly factor for complex IV
in Saccharomyces cerevisiae and its human homolog is SURF1, and is the most frequent cause
of Leigh syndrome (Rossi). SHY1 also plays a role in the insertion of Haem a, an essential co-
3. 2
factor for cytochrome c oxidase,however it is still unclear whether SHY1 acts as a chaperone
protein or has a direct role in the insertion of Haem.
Cytochrome c oxidase deficiencies are one of the most common defects of the respiratory
chain in mitochondrial diseases (Li). Cytochrome c oxidase deficiency affects several parts of the
body including the muscles, heart, brain, and liver (Robinson). Signs of cytochrome c oxidase
deficiency are apparent in the early stages of life. If it results in the onset of Leigh syndrome,
severe problems such as the loss of mental function, movement problems, cardiomyopathy, and
brain abnormalities (Robinson).
SHY1 is a mitochondrial inner membrane protein that is essential for the assembly of
complex IV. The protein associates with complex IV intermediates and complex III/complex IV
supercomplexes (Mick). Specifically, SHY1 is required for the efficient assembly of cytochrome
c oxidase, aka complex IV. SHY1 interacts with Mss51-COX14 in a step that couples the
regulation of COX1 in the primary stages of cytochrome c oxidase (Mick).
In this experiment, mutations were intentionally created in the SHY1 deletion strain of
Saccharomyces cerevisiae, using MnCl2 that would restore the assembly of the cytochrome c
oxidase complex. The results showed that the mutated yeast were able to undergo aerobic
respiration and grow despite the absence of SHY1. This is indicative of the fact that the yeast
were able to overcome the mutation and grow normally.
4. 3
Methods
Mutations: 10 micromolar of Mn+2 will accumulate in yeast cells. The cells were grown on
glucose YPD media. MnCl2 was then added to the cells and were incubated for 16 hours, shaking
at 250rpm at 30ºC. Then, cells were placed on the selective EG media and grown at 30ºC until
colonies appeared.
Large-scale Culture for the Growth and DNA Tests: 1 mL overnight culture was transferred into
a fresh 50 mL YPD sterile flask. It was incubated at 30oC for 3 hours to grow, while spinning at
250 rpm. Then, 4 mM MnCl2 was added after the incubation period.
Growth test with serial dilutions: For each strain, 10 mL of YPD culture was grown and the
absorbance was measured at A600. Several dilutions were made to obtain an absorbance value of
1. Sterile water was used to accomplish this. Then, 4 serial dilutions were prepared with 450
microliter H2O and 50 microliter from the previous dilution. 3 microliter of each dilution were
plated on one YPD plate and on a YPEG plate. The plates were then incubated for 3 days at 30oC
to grow.
Small-scale total DNA preparation: Cells were grown overnight in 10 mL YPD. They were then
centrifuged at 3000 rpm for 10 minutes. The supernatants were then removed and washed with
10 mL 1.2 M Sorbitol. Then, at the same speed, the cells were spun again and the supernatant
was removed. The pellets were suspended again using 0.8 mL Zymolyase buffer (ingredients).
Then, the cells followed a 1 hour incubation period at 37oC. After the incubation period, 10 mL
5. 4
1.2M sorbitol was added. Then, they were centrifuged at 1000 rpm for 10 minutes. Afterwards,
0.8 mL 1% SDS was added. Then, 0.8 mL phenol was added for protein denaturation. Then, they
were centrifuged for 5 minutes at 4000 rpm. Then, 0.6 mL of the upper aqueous phase was
transferred to to a new set of Eppendorf tubes, where they were washed down with 2 mL ether to
get rid of the phenol. Then, 50 uL 5M NaCl and 1.8mL ethanol was added. The tubes were then
centrifuged for a period of 10 minutes at 4000 rpm. After centrifuging, the tubes were rinsed
using 80% ETOH. Then, the tubes were centrifuged for a period of 10 minutes again at 4000
rpm. The supernatant was then removed and were left to dry. Then, they were dissolved in 200
microliter of 10 mM Tris pH 7.5, followed by the addition of 2 uL RNase and then incubated at
37˚C for 15 minutes. Then, 200 uL of phenol was added. The upper layer was then transferred to
a tube. It was washed down twice using ether. Thereafter, 10 uL 5M NaCl was added along with
180 uL ETOH. The tubes were then centrifuged for a period of 10 minutes at 4000 rpm. After
centrifugation, the tubes were rinsed twice using 80% ETOH, and were then left to dry. A week
later, 15 microliter H2O was added to the DNA sample. Finally, the nucleic acid to protein ratio
was determined to be greater than 1.8.
PCR: 35.75 uL H2O was added to the tubes. Then 10 uL buffer was added. Then, 1 uL dNTPs
was added. Next, 1 uL of forward primer was added. The addition of 1 microliter reverse primer
followed. Then, 1 uL DNA template was added. Finally, 0.25 microliter DNA polymerase was
added. It was mixed by pipetting. The DNA was was then placed in a thermal cycler and was
ready for PCR. The DNA was heated to 95˚C, denaturing it, so the strands would separate. Then,
the temperature was cooled down to 55˚C for annealing, so that the primers could bind to to the
6. 5
template DNA by base-pairing. Then, the temperature was heated to 72˚C for extension for 1
minute. The PCR steps were repeated a total 35 times.
Gel Preparation: 10 uL 5X loading dye was added to 50 uL of DNA (10 microliter + 50
microliter). 0.5 g of Agarose was added to the flask, followed by the addition of 40 mL TAE
buffer. It was then heated in a microwave until the Agarose dissolved. After taking it out from
the microwave, it was cooled by washing it with cold water. Thereafter,2.0 microliter Fluorescent
DNA was added to stain the Agarose solution, and was set down. The samples were then loaded.
Afterwards,the sample was visualized under UV light. The DNA band was then cut out and placed in the
tubes.
Extraction of DNA froma Gel Source: 3 volumes of binding buffer (300 uL per 100 mg Gel) was added.
The tubes were then heated at a temperature of 55˚C until the gel melted. Then, 5uL of silica powder
suspension was added. The tubes were then incubated for a period of 5 minutes at 55˚C; it was required to
mix occasionally. The tubes were spun to pellet the silica (DNA) at maximum speed for 30 seconds. The
supernatant was then removed. Then, 500 uL ice cold washing buffer (ethanol) was added. Then, using a
vortex and a pipet, it was resuspended by to drive the silica into suspension. The tubes were then
centrifuged again and supernatant was removed afterwards. This process repeated three times. The tubes
were then left to dry until ethanol was removed. Then, the tubes were resuspended in 10 μL H2O
(elution). Then, they were incubated for 5 minutes at a temperature of 55˚C. The tubes were then spun
and the supernatant was retained and transferred to another tube. Then, the DNA was quantitated using a
Nanodrop, which measures how much DNA is in a solution.
12. 11
Yeast was grown from individual colonies grown on the plate seen in figure 2. The
nanodrop reading for the yeast sample shows that the DNA was low in concentration. However,
the DNA that came back from sequencing showed a 97% match with the wild type, as indicated
in the results above. The main focus of the experiment was to see if yeast were able to grow in
the absence of the SHY1 gene after mitochondrial DNA had been mutated. Table 1 shows that
the absorbances of the �SHY1 strains are quite high, not too far from those of the wild type,
W303. Table 3 shows that �SHY1 strains were positive for growth after most experiments were
performed. Furthermore, figure 5 shows the growth of different strains of �SHY1 yeast
compared to that of wild type W303, and they all showed healthy growth as well. Figure 1 shows
the growth of mutated �SHY1 yeast on EG plates compared to growth on YPD plates. What this
suggests is that the� SHY1 yeast are able to perform aerobic respiration despite undergoing
mutations to a gene essential for the assembly of cytochrome c oxidase. It may be possible that
fermentation is occurring in the event that aerobic respiration cannot be performed, however, this
is less likely because there would be a noticeable difference in the growth of the yeast compared
to the wildtype, if this were indeed the case. Once more referring to figure 5, it is evident that at
higher concentrations, individual colonies are formed, this indicates that these yeast were
growing well after having been mutated. After looking at all the available data, it is say to say
that �SHY1 strains of yeast are able to grow properly on both YPD and EG plates.
What this would suggest, then, is that human beings might also be able to perform
aerobic respiration if the SURF1 gene was mutated as well. The SURF1 gene in humans is the
homolog of the yeast SHY1 gene (Mashkevich). This can be seen in figures 6 and 7 above.
13. 12
Again, this would seem to suggest similar versatility of the SURF1 gene in humans and lead to
aerobic respiration occurring unhindered, however, we know this isn't the case. Research shows
that leigh syndrome, the result of mutations in complex IV assembly genes, is especially linked
to mutations in SURF1 (Lee). Leigh syndrome is proof that mutations in cytochrome c oxidase
assembly are not benign and are not easily fixed by the body (Lee). This could possibly suggest
that yeast mutations are not truly indicative of the result of mutation in their human homologs.
This discrepancy could possibly be an area of further study.
14. 13
References
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(2012). SURF1‐ associated leigh syndrome: A case series and novel mutations. Human mutation, 33(8),
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