20.109(S07): Screen for phenotypes, isolate RNA

Introduction

Life is evidently more complicated than base-pair chemistry since even a perfect cataloging of an organism’s genetic information does not allow us to build a cell from scratch. Furthermore, two cells in a multicellular organism can have identical genomes but different physiologies. Even at an organismal level, we see that identical twins do not exhibit identical traits. Moreover, minimal differences in DNA code can separate species. With human DNA sequence less than 1.25% different from that of chimpanzees, it appears too simple to believe, “…our fate is in our genes" (a phrase that was popularized when Nobelist Jim Watson told TIME magazine in 1989, "We used to believe our destiny was in the stars; now we know in large measure our fate is in our genes.”)

Rather than genetic content it may be changing expression patterns of a genome that explain cellular differentiation and development. A single cell develops into a differentiated multicellular organism by varying gene expression in groups of cells as they divide. A liver cell must express the parts of the genome related to liver function, while a skin cell uses the parts of the code for making skin related proteins. Scientists are trying to describe other “-omes,” such as the “transcriptome” (the complete RNA content of a cell or organism) and the “proteome” (its total protein content), to complement the cataloging of an organism’s total DNA content. Fortunately, techniques for detecting the RNA and protein output of a cell abound. Older methods have been used productively for decades and newer techniques offer increased sensitivity and higher throughput methods. Because of their widespread use, several fundamental techniques in gene expression analysis will be considered in detail.

One classic technique for monitoring gene expression is Northern analysis. In this approach, an RNA sample is electrophoresed through a polyacrylamide matrix and then transferred (“blotted”) from the gel to a solid support, usually made of nitrocellulose or nylon. The blot is then probed with radiolabelled DNA, or less often RNA, and then exposed to X-ray film. Hybridization of the probe to the blot is detected as a darkened area of the film and the signal gives information about the size and concentration of that RNA in the original sample. Valid Northern analysis data includes re-probing the blot with a “loading control” to demonstrate that each sample was equally loaded on the agarose gel and evenly transferred to the blot. Typical loading controls are 18S rRNA and actin mRNA since these are abundant transcripts in most cell types and are seldom affected by experimental conditions.

Sample Northern gel

An alternative to Northern analysis is q-PCR (quantitative-Polymerase Chain Reaction, sometimes also called RT-PCR which can stand for either Reverse Transcriptase-PCR or real time-PCR ... RT RT-PCR??). You gained experience with “end point PCR” at the start of this experiemental module when you used the final product of that amplification to transform yeast. With q-PCR, quantitative information is gleaned from the early stages of the PCR cycling protocol. A specialized thermal cycler is used as well as a fluorescent dye to monitor the amount of double stranded DNA in each reaction at each step of the PCR protocol. RNA is isolated from cells of interest (as you will do today) and converted to DNA using an enzyme called reverse transcriptase (as you will do next time). This DNA serves as the template in the q-PCR reactions. After a limited number of PCR cycles, the amount of PCR product can be sensitively detected by its fluorescence and quantitatively reflects the amount of transcript in the original sample.

A third means of measuring RNA in a cell is through a DNA microarray. In this technique the RNA from two cell types are simultaneously hybridized to DNA probes on a slide. The DNA probes represent some or all of the genome, enabling the relative amounts of gene expression for each gene in both cells to be assessed. This is the technique you will perform next time, and more details will be presented then.

Today we will experimentally address two related questions. First, we'll ask if losing a SAGA-subunit changes the yeast's physiology, looking for phenotypes associated with the mutation you've directed into the yeast genome. Phenotypic differences may reveal aspects of gene regulation and protein function related to the SAGA-complex and for the subunit you've chosen to study. Second, we'll consider each gene that's expressed in the wild type and the mutated cells, isolating RNA today so next time you might identify mRNAs whose production is affected when the SAGA-subunit you've selected is deleted. Both experiments will be informative, but it's important to note that neither can determine if, mechanistically speaking, the observed changes are a direct or indirect consequence of the mutated gene.

Protocols

Part 1: Agarose gel of PCR products

You will share an agarose gel with one other group.

1. Retrieve your PCR samples from the teaching faculty.
2. Move 10 ul of each sample to a labeled eppendorf tube.
3. Add 2 ul of loading dye to each of the eppendorf tubes.
4. Load these aliquots onto a 1% Agarose Gel (1xTAE), according to the following table.

Lane	Sample	Volume to load
1	100 bp Marker	5 ul
2	GROUP 1: PCR product/FY2068 template	~12ul
3	GROUP 1: PCR product/candidate A	~12ul
4	GROUP 1: PCR product/candidate B	~12 ul
5	GROUP 1: PCR product/candidate C	~12 ul
6	100 bp Marker	5 ul
7	GROUP 2: PCR product/FY2068 template	~12ul
8	GROUP 2: PCR product/candidate A	~12ul
9	GROUP 2: PCR product/candidate B	~12 ul
10	GROUP 2: PCR product/candidate C	~12 ul

The gel will run at 125V for approximately 45 minutes, and one of the teaching faculty will photograph it for you.

Part 2: Spot test candidates for phenotypes

The yeast you are working with, S. cerevisiae, was the first eukaryotic organism to have its genome fully sequenced.Goffeau et al, Science 1996 A full 10 years later there is still ongoing discussion and work on how to best annotate the genome. Fish et al, Yeast 2006 There is general agreement that S. cerevisiae genes number ~6000, and of these approximately 20% are essential. The essential genes also show noteable conservation, with homologs identified in other sequenced organisms. Those essential genes that have no human homologs are useful antifungal drug targets. How to best make sense of the other 4000 annotated genes? About 15% of these affect the cell's physiology in at least one of several discernable ways. In many cases, the function of these genes can be revealed by the phenotype that arises from their loss.

Many phenotypes have been described for S. cerevisiae. A very fine review of these was published by Michael Hampsey in Yeast 1997 He groups the phenotypes into the following categories:

Category	example of phenotype	Assay
Conditional growth	e.g. TS (37°) or CS (14°)	growth on rich media at restrictive condition
Cell cycle defect	e.g. large, unbudded cells or very few unbudded cells	microscopic examination
Mating and sporulation defect	e.g. failure to produce mating factor	e.g. halo assay
Auxotrophies, carbon catabolite repression, nitrogen utilization	e.g. Snf phenotype (Snf = "sucrose non-fermenter")	growth on incomplete or alternative media (e.g. other sugars)
Cell morphology and wall defect	e.g. bud localization	e.g. calcofluor staining
Stress response defect	e.g. sensitivity to heat shock	e.g. incubate 1 hr at 55° then test for viability
Sensitivities	e.g. canavanine	growth in presence of analog, antibiotic or drug
Carbohydrate and lipid biosynthesis defects	e.g. nystatin	growth in presence of synthesis inhibitor
Nucleic acid metabolism defects	e.g. UV light sensitivity	growth in presence of damaging agent
Other	e.g. caffeine	growth in presence of...

You should begin by looking at the Hampsey review to familiarize yourself with the range of phenotypes that are detectable and the kinds of things each can tell you about the yeast displaying them. Next, consider the kinds of processes you might expect to be affected in your mutant. Do you know what phenotypes SAGA-mutants show? Do you know what processes the subunit you've deleted might play into? You do not have to know what the outcome will be to test for growth on a particular medium. It's certainly possible that no one has ever tested your strains under the conditions that you'll try. In an ideal world you would have limitless time and resources and you might explore all the possible phenotypes, individually and in combination (looking for a failure to grow on galactose as the sole carbon source when incubated at 37° for example). In reality, we have prepared some media for you to try today. You are welcome to use the plates we have and the incubators in the lab and if there are particular other experiments you'd like to try, please ask the teaching faculty and we will do our best to arrange things.

To test the phenotypes of your deletion strains you will perform a common lab technique, namely a "spot test." In this assay, overnight cultures are serially diluted in 96 well dishes and a few microliters of each dilution is spotted using a multichanel pipetman onto the surfaces of some petri dishes, each strain in a different row and each serial dilution in a different column. The resulting data may look like:

YPD

YPD + 3%formamide

where the image on the left shows all the strains being compared, grown on rich media whereas the image on the right reveals the sensitivity of the same strains to growth on rich media plus 3% formamide. Strains #2, #3, #5 and #6 clearly grow less well on formamide-containing media since the spot at which the cells are no longer detected moves from the 10^-6 dilution back to the 10^-4 or 10^-1 dilution, depending on the strain.

1. Decide on the types of media you will examine and any growth conditions (temperature etc). Retrieve these plates and label them with the date, your team color and the strains you are examining.
2. Vortex the strains that you innoculated last time: FY2068, candidate A, candidate B, and candidate C.
3. Next move 100 ul to the first well of a 96 well dish: FY2068 to position A1, candidate A to B1, candidate B to C1 and candidate C to D1.
4. Add 90 ul of sterile water to the wells A2-A6, B2-B6, C2-C6 and D2-D6.
5. Using your P20 set to 10 ul, pipet the yeast in well A1 up and down to mix them then move 10 ul of the yeast into the water that is in A2. Repeat, moving 10 ul of the dilution from A2 to A3 then 10 ul from A3 to A4 all the way out to A6.
6. Repeat the serial dilution series for the yeast in wells B1, C1 and D1.
7. Lay your labelled petri dishes open on the bench top.
8. Place 4 pipet tips on the multichanel pipetman and spot 3 ul from column 1 onto the leftmost side of each petri dish. You can use the same pipet tips for all your plates but be sure that the tips are properly filling each time. Sometimes liquid can accumulate in them to give errors in measurement.
9. Change pipet tips and then spot 3 ul from column 2 next to the spots you just placed from column 1. Spot all the petri dishes in this same way.
10. Repeat for columns 3-6.
11. Carefully replace the covers on the petri dishes but do not move them from your bench until all the spots have dried.
12. Wrap them in your colored tape and place them in the incubators.

Part 3: Isolate RNA

Once you know which, if any of your candidates have the SAGA-subunit deletion, proceed to isolate RNA from that strain and from the parent strain, FY2068.

Measure cell number

Microarray analysis allows us to compare the population of RNA from two samples with an identical number of cells, namely 2 x 10^7. Begin this part of today’s lab by measuring the density of your overnight cultures then converting this spectrophotometric measurement into cell number.

1. Make a 1:10 dilution (100 μl into 900 μl water) of each culture.
2. Use 0.5 ml to measure the optical density at 600 nm for each dilution, using water to blank the spectrophotometer.

	OD600nm	Cells/ml (using 5 OD600~108 cells/ml)
FY2068
SAGA mutant

Spheroplast

1. Collect 2 x 10^7 cells of each strain (on the order of 200 ul if the strains are both densely grown) in eppendorf tubes
2. Harvest by spinning the tubes in a microfuge, full speed, 1 minute.
3. Aspirate to remove all the supernatant.
4. Add 1 ml of supplemented Y1, resuspend the pellet, and incubate at 30° taped to the roller drum rolling at speed 4 for 15 minutes. Y1 is supplemented with zymolyase, an enzyme that will break down the yeast cell wall.
5. Microfuge your samples at 1.8 rpm = 300 rcf for 5 minutes.
6. Aspirate to remove all the supernatant from each sample. You can spin 1 minute more at 1.8 rpm to spin the last bits of liquid off the walls of the tubes and then aspirate or use a pipetman to remove.

RNA prep

RNA is strikingly different from DNA in its stability. Consequently it is more difficult to work with RNA in the lab. It is not the techniques themselves that are difficult; indeed, many of the manipulations will seem identical to those used for DNA. However, RNA is rapidly and easily degraded by RNases that exist everywhere. There are several rules for working with RNA. They will improve your chances of success. Please follow them all.

• Use warm water on a paper towel to wash lab equipment, like microfuges, before you begin your experiment. Then wipe them down with “RNase-away” solution.
• Wear gloves when you are touching anything that will touch your RNA.
• Change your gloves often.
• Before you begin your experiment clean your work area, removing all clutter. Wipe down the benchtop with warm water then “RNase-away,” and then lay down a fresh piece of benchpaper.
• Use RNA-dedicated solutions and if possible RNA-dedicated pipetmen.
• Start a new box of pipet tips and label their lid “RNA ONLY.”

You will isolate RNA from your yeast samples using the Qiagen RNA-easy miniprep kit.

1. Add 350 ul RLT+BME to each pellet. Pipet up and down to resuspend then vortex one minute to lyse spheroplasts. You can spin the samples at this point for 2 minutes if there is any insoluble material.
2. Add one volume of 70% EtOH. Pipet up and down immediately upon addition and apply sample to the spin column and collection tube that you can collect from the teaching faculty.
3. Microfuge 30 seconds at full speed.
4. Discard the flow-through into the sink or a conical tube that you set up on your bench for collecting waste. Save and re-use the collection tube for the next step.
5. Wash the column with 700 ul RW1. Microfuge 30 seconds at full speed.
6. Discard the flow-through and the collection tube. Get a new collection tube from the teaching faculty.
7. Wash the column with 500 ul RPE. Microfuge 30 seconds at full speed.
8. Discard the flow-through but re-use the collection tube.
9. Microfuge the column for 1 minute full speed to dry it.
10. Move the column to an RNase-free eppendorf tube with the cap cut off.
11. Elute the RNA from the column by adding 30 ul of RNase-free water directly to the membrane.
12. Allow the water to remain on the membrane for one minute, then microfuge, full speed for 1 minute.
13. Repeat with a second 30 ul aliquot of water, so each sample will yield approximately 60 ul of RNA.

Measure RNA concentration

1. Measure the concentration of your RNA sample by adding 5 μl to 495 μl sterile water. The water does not have to be RNase-free since the RNA can be degraded and still give legitimate readings in the spectrophotometer. Make your dilutions in an eppendorf tube and use your P1000 to transfer the dilution to a quartz cuvette. Measure the absorbance at 260 nm. Water in one of the optically paired cuvettes should be used to blank the spectrophotometer, but if another group has done this already, it does not have to be repeated.
   • A few things to be aware of when using quartz cuvettes:
     • They are very expensive.
     • The lab has only one set.
     • When you are done using the cuvette, you should carefully clean it by shaking out the contents into the sink and rinsing it once with 70% EtOH, then two times with water. Quartz cuvettes get most of their chips and cracks when someone is shaking out the contents since it is so easy for the cuvette to slip from wet fingers or be hit against the sink. Don’t let this happen to you.
2. To determine the concentration of RNA in your sample, use the fact that 40 μg/ml of RNA will give a reading of 1 A260.

RNA Sample	A260	Conc of dilute RNA	Conc of undiluted RNA
FY2068
SAGA mutant

DONE!

For next time

Please print out and hand in next time.

1. Calculate the volume for 2 ug, 1 ug and 0.5 ug of each RNA sample that you prepared. Show your work.
2. Prepare a figure for the agarose gel you ran to check by PCR for your deletion. The gels are posted on the discussion page. Be sure the figure has a title (usually the conclusion you'd like the reader to draw), and a short description of what was done (in past tense) and what's shown (in present tense). You should number the lanes for clarity and refer to those numbers in the description. You should also indicate the sizes of the molecular weight markers.
3. In some cases, mutant phenotypes are a direct consequence of the mutated gene and in other cases, phenotypes arise indirectly. For a hypothetical gene ("YFG1"), please describe a case when the phenotype is a direct consequence of a yfg1 mutation and a case when it's an indirect consequence. Finally describe an experiemnt you could do to determine the question of direct vs indirect effects.

Reagents list

• Supplemented Y1: 1M sorbitol, 0.1M EDTA + zymolyase (10 units/ml)

• YPGal+antimycin A
  • 10g yeast extract
  • 20g peptone
  • 20g agar
  • add 950ml water, autoclave 20min, stir to cool
  • add 50ml 40% sterile filtered galactose + 1ml of 1mg/ml Antimycin A when cooled

• YPG
  • 10g yeast extract
  • 20g peptone
  • 20g agar
  • add 970ml water, autoclave 20min, stir to cool (cool to 65C)
  • add 30ml sterile 100% glycerol

• YPAc

• YP+3%form
  • In one 2L flask: 10g yeast extract, 20g peptone, 460ml water
  • In another 2L flask: 20g agar, 450ml water
  • autoclave both flasks 20min, stir to cool
  • add 10ml TRP + 30ml formamide + 50ml 40% sterile glucose to first flask
  • combine contents of flasks

• SC-lys

• YP +rapamycin

next year: no Ac, asked for cyclohexamide, asked for 3AT. Need better labeling system for plates.