Control of Gene Expression in Yeast: Heat Shock Promoters: Report.

Transcriptional Regulation Due to Environmental Stresses

Eukaryotic and prokaryotic cells react to environmental stimuli by activating the expression of certain genes, which encode protein, enzymes that will help the cell endure the environmental change. Examples of environmental stimuli include Heat shock factor, which expresses genes for survival at high temperatures, hypoxia inducible factor, which expresses genes for survival in low oxygen conditions, and sterol regulatory element binding protein, which maintains proper lipid levels within the cell.

Heat Shock in Eukaryotes and Their Response

This experiment aims to study the effect of gene expression in a cell responding to high temperature stress using heat shock promoters. Heat shock gene expression is activated as a response to increased temperature. Increasing the temperature activates the transcription of the cell’s multiple heat shock genes. The proteins synthesised help reduce the damage from thermal denaturing of important cellular proteins. A large number of proteins is formed rapidly in a response to heat shock which act as ‘chaperones’ aiding other proteins to retain their 3 dimensional form and function properly. The heat shock genes are activated by the binding of the protein called heat shock transcription factor to the heat shock response element. The heat shock transcription factor is not active in non-heated cells, however increasing temperature cause a structural change of the protein that allows it to bind to the heat shock response element in DNA. Then the protein is modified by phosphorylation, allowing it to express gene transcription. (Hardin, 2012)

Reporter Genes

A reporter gene is a gene that is attached to a regulatory sequence of another gene that’s being studied, in this case, in a cell culture. A gene is chosen as a reporter because its characteristics that are expressed on an organism are easy to identify and measure or because they are selectable markers. Selectable markers are genes introduced into a cell that grants it a specific characteristic for artificial selection. Reporter genes are usually used to specify whether a specific gene has been expressed or taken up by the cell. In this case, the promoter hsp26 gene from yeast has been attached, artificially, to a gene that’s coding for the enzyme Beta-galactosidase. Beta-galactosidase is an enzyme present in bacteria and its function is to breakdown Beta-galactoside sugars. For example, lactose is converted to galactose and glucose. Replacing the lactose with the substrate o-nytrophenyl-beta-galactoside (ONPG), instead results in the formation of galactose and o-nitrophenol (ONP) – a yellow discolouration which is due to high pH. Hence, the gene for Beta-galactosidase, named LacZ, has an easily assayable product. LacZ is the reporter gene, as the promoter is Beta- galactosidase, whose activity is being measured. 2

The strain of yeast (Saccharomyces cerevisiae) being used is W303. It contains a plasmid (pUKC414) which consists of the promoter; hsp26, and the Beta- galactosidase coding sequence. Approximately, one copy per cell was present in each plasmid.

In this particular experiment, placing the yeast under heat shock conditions (30-45 degrees Celsius) caused the hsp26 promoter to activate hsp26 and Beta-galactosidase. Beta-galactosidase converts lactose into galactose and glucose. Beta-galactosidase also hydrolyses the ONPG molecule into galactose and o-nitrophenol (ONP). The end solution was yellow, due to the presence of ONP, which was used to inspect enzyme activity using a colorimetric assay. 1

The hypothesis was that as temperature increases, the intensity of yellow colour increases as ONP presence increases as it tries to protect the cell, hence B-galactosidase activity is increased. The null hypothesis was that increase in temperature would have no effect on the B-galactosidase activity, no matter what incubation time was.Did you know that you can transfer from the second-year of a Science degree into the third year of medicine? Check out graduate-entry medicine in Europe.

Method

The protocol provided in the in-lab hand-out was used, however one modification was intentionally made. The first incubation time was changed from 15 to 20 minutes to allow more time for B-galactosidase to convert the disaccharides, producing more ONP. This didn’t seem like enough time because only the 39 deg. Celsius tube had a very tinted yellow colour. All the tubes were returned to the water baths for an extra 5 minutes. Furthermore, too much SDS was accidentally added, 200 instead of 20ul, to one of the tubes, however this was assumed to have no significant impact on the results. This error was ignored. Some spillage occurred with tube 37 deg Celsius however there was nothing that could have been done to fix this as it was too late to start the process again.

Results

The specimen results were used because the individual results seemed very random and chaotic. Individual results are shown below but they were regarded as an anomaly and were assumed to be inaccurate.

The specimen results were examined and the Beta-galactosidase activity was calculated. Beta-galactosidase activity is measured by working out ONP (nmol) /cells assayed (ml)/time left in bath (mins). The absorbance was converted into nmol via the following equation;

0.0045 at 420nm = 1 nmol of ONP, which was then divided by volume and time left in water bath (15 rather than 25mins, because specimen results were used). Hence, B-galactosidase activity was calculated for all the results. An example of the first calculation is given below:

B-galactosidase activity (nmol/ml of cells/min) : ( ({0.247⁄0.0045}⁄1.5))⁄15

=2.440 nmol/ml of cells/min

The following table shows Absorbance for both incubation temperatures and the calculated activity of B-galactosidase, for both time durations.

Absorbance at 420nm B-galactoside Acitivty (nmol/ml cells/min)

Temp

(deg. C) 20 mins 45 mins 20 mins 45 mins

30 0.247 0.235 2.440 2.321

37 0.330 0.556 3.259 5.491

39 0.313 0.924 3.091 9.126

42 0.208 0.254 2.054 2.509

45 0.242 0.153 2.390 1.511Did you know that you can transfer from the third-year of a Science degree into the fourth year of medicine? Check out graduate-entry medicine in Europe.

Discussion

Hsp26 gene from yeast was an artificially attached gene whose purpose was to code for the enzyme Beta-galactosidase which eventually lead to the production of ONP, the yellow colour being measured. This was introduced into the cell via Plasmids, instead of modifying a gene in the yeast chromosomes, because plasmids are easier to manipulate.

The results showed a sensible pattern. When the tubes were incubated for a longer period, 45 minutes, optimal enzyme activity was shown at 37-39°C. However, enzyme activity dropped significantly afterwards from 9.1 at 39°C to 2.5 nmol/ml of cells/min at 42°C, which could be due to the enzymes not being able to cope with the heat shock anymore as they begin to denature. At 30°C, not many enzymes were active as the temperature could be considered moderate or low.

The hypothesis was fairly true since the B-galactosidase activity did increase as temperature increased however to a certain point. After 42°C, activity dropped significantly for both incubation times. It was also found that the longer the incubation the faster the activity rate of B-galactosidase. The null hypothesis was regarded as false.

B-galactosidase activity was highest for the cells at 37°C as peak activity was reached, for the 20 min incubation. However, peak activity for incubation of 45 mins was at 39°C. This is above the yeast cells’ optimum growth temperature; 37°C, which makes sense for heat shock proteins to be very active providing the cell with thermotolerance, as the proteins have more time (45 compared to 20 mins) to be produced.

When the tubes were incubated for only 20 minutes, a fairly similar pattern occurred however at a much slower rate. Optimal temperatures were also at 37-39 with a B-galactosidase rate ~3 nmol/ml of cells/min. This could be due to the heat shock response being more receptive when allowed a longer incubation time period, so all the circular DNA plasmids having more time to penetrate the yeast cell membranes.

The heat shock response was also active at 30°C which is surprising since there’s no high temperature that the cell is competing with; however this is normal due to the increase in temperature during incubation. Usually there is some heat shock activity in the cell due to a the presence of a small number of heat shock proteins that exist in the cell to cope with other functions of repair, altering protein confirmation, modulating protein activity, modifying protein degradation and folding peptide chains correctly during protein translation. (Takayama, 2003)

This study could be improved by measuring enzyme activity at a more precise range of temperatures and different incubation times to find the optimum B-galatosidase activity as each strain of yeast performs differently at each individual temperature. However the cells cannot be kept in heat shock for too little time or too long, as the cells need a recuperation period to recover but also need time to replicate the plasmid DNA while incubated, and produce the proteins required for heat shock.

Furthermore, it’s predicted that the individual experiment failed to produce any sensible results due to several factors. Some of which could be due to inconsistencies in the age of the yeast cells being used, amount of circular plasmid DNA used, the duration and intensity of the heat shock and the time allowed for recovery period. These factors could be taken into account if the experiment was to be repeated to produce a more accurate result. Some complex factors that cannot be controlled in cells include the stability of mRNA and other factors that affect the expression of the promoter being studied; hsp26.

In conclusion, this experiment showed the heat shock factor was an important natural phenomenon that helps the cell cope with one of its many environmental stresses. It is essential for life, and its complex activity on a molecular level needs to be studied in more detail to determine accurate results in how far the cell’s natural state can be stretched while still functioning with no complications. Artificially introducing the promoter hsp26 to the plasmid showed that it was an essential factor that lead to the production of B-galactosidase, which lead to the production of ONP – the yellow product that was measured, indicating a physical indicator of activity of heat shock proteins.

The full report with tables and graphs by a graduate from the University of Huddersfield is available for free, however first we ask you to like our facebook page and then request it via messenger and we’ll happily send it to you.


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