Cells in Space
Cell Biology and DNA Microarrays
by Lesli Adler, Montgomery Public School District, MD
In this simulation activity, students will investigate the use of a new procedure that connects
molecular biology, cell biology, bioethics, and technology. DNA microarrays are powerful tools used
to measure the activity of cells as they transcribe their genes into proteins that control cellular
biochemical reactions and giving cells their unique functions.
There are several biological and technology concepts highlighted in this activity:
 Genes differentially express (transcribe into mRNA) depending on the conditions under which
they are grown. They may express, express somewhat, or not at all. This differential
expression leads to the phenotype.
 All cells of the organism contain the same genes; all organisms of the same species contain
the same genes, although some may have variations in nucleotide sequence leading to
differential expression, including dominant and recessive conditions.
 Cells acquire energy through anaerobic or aerobic cellular respiration. Transcribed proteins
and enzymes are responsible for these biochemical processes.
 Nucleic acids (DNA, mRNA, cDNA) can complementary bind and this property can be used in
a powerful new technology
 Microarrays can be used to measure differential gene expression and that technology can
have powerful consequences on the ability of researchers to understand basic life processes,
and have profound consequences on society
MSDE Standards: http://www.mdk12.org/instruction/curriculum/science/index.html
Goal 1: Skills and Processes: The student will demonstrate ways of thinking and acting inherent in
the practice of science. The student will use the language and instruments of science to collect,
organize, interpret, calculate, and communicate information
Expectation 1.7: The student will show that connections exist both within the various fields of
science and among science and other disciplines including mathematics, social studies, language
arts, fine arts, and technology
Indicator 1.7.1: The student will apply the skills, processes, and concepts of biology, chemistry,
physics, and earth science to societal issues
Indicator 1.7.2: The student will identify and evaluate the impact of scientific ideas and/or
advancements in technology on society
Indicator 1.7.6: The student will explain how development of scientific knowledge leads to the
creation of new technology and how technological advances allow for additional scientific
accomplishments
Goal 3 Concepts of Biology: The student will demonstrate the ability to use scientific skills and
processes (Core Learning Goal 1) and major biological concepts to explain the uniqueness and
interdependence of living organisms, their interactions with the environment, and the continuation of
life on earth.
Expectation 3.2: The student will demonstrate an understanding that all organisms are composed of
cells which can function independently or as part of multicellular organisms
Indicator 3.2.1: The student will explain processes and the function of related structures found in
unicellular and multicellular organisms
Indicator 3.2.2: The student will conclude that cells exist within a narrow range of environmental
conditions and changes to that environment, either naturally occurring or induced, may cause
changes in the metabolic activity of the cell or organism
Schedule: This activity is intended to be used incorporated into the unit on cellular respiration and will
connect the molecular genetics of biochemical activities of cellular respiration to the use of a new
technology.
Day 1 & 2:
Pretest; PreTest Answers: 1.a, 2.d, 3. yellow, 4. a & b
 Introduce the activity and the scenario. Review pertinent biochemical processes of cellular
respiration.
Reference: http://www.spaceref.com/news/viewpr.html?pid=13508
 Perform computer microarray simulation
Reference: http://www.bio.davidson.edu/Courses/genomics/chip/chip.html.
 Perform paper microarray activity
Reference:
1.
2004 Carolyn A. Zanta. UIUC-HHMI Biotechnology Educarion and Outreach
Program (BOEP), www.lifw.uiuc.edu/hughes/footlocker
2.
Kathleen Gabric, 2005
Day 3:
 Perform lab activity
 Discuss class results
 Answer pertinent written questions
Day 4:
 Perform extension activity and answer pertinent questions
Reference:
1. Preferred, but by subscription only: Time magazine, January 11, 1999 Vol. 153 No. 1;
Gorman, Christine. The Future of Medicine; Drugs By Design. Subscription purchase.
http://www.time.com/time/archive/preview/0,10987,989981,00.html
Day 5:
 Post test and closing discussion; PostTest Answers: 1.a, 2.d, 3. yellow, 4. a & c
Notes:
The probe solutions (ES1, EF1, EB1), which are made of various pH indicators, are embedded in
agarose which must remain melted until spotted onto the slide. Once dropped onto the slide, they will
harden and adhere to the slide. They must be handled carefully.
Remind students to be careful when placing the slide into the cell solution in the slide developing tray.
Blue plastic slide boxes work very well for this. Students should receive enough A solution to fill the
box so it covers the adhered spots. This is mild NaOH. Students should wear goggles and gloves.
We can send reagents in bulk so you can “mix and match” and custom design your class results. In
this way some yeast strains can appear to be more effective than others. The reagents will be
designed to the following color key: If the spot is red, then gene F1 is transcribing, blue, B1 is
transcribing. Purple if both are transcribing (S1). Remember- the students are tagging all mRNA
produced in aerobic (red) vs anaerobic (blue) conditions.
Ordinary microscope slides can be used for this. Gently roughen the surface of the slide so the
agarose adheres better. A sample real microarray slide will be sent with the kit. Additional
microarrays can be obtained through various companies that supply microarray equipment, such as
www.affymetrix.com
Pre Test
Cells in Space
1. Which of the following describes a DNA microarray:
a. a new technology that allows scientists to view real time gene expression
b. a method of isolating DNA so it can be used for gel electrophoresis
c. the form in which DNA is packaged into chromosomes
d. the graphic results of a forensic DNA sequencing profile
2. DNA arrays can be used for all of the following except:
a. identify whether someone has a certain disease
b. tell whether the genes for living in anaerobic conditions are transcribing
c. genetic ID card
d. predict whether someone will die of heart disease
3. The following diagram displays the results of a particular DNA array: Which color spot(s) indicate
that all of the target genes are all transcribing:
O
O
O
O
O
RED GREEN YELLOW BLACK RED
O
O
O
O
O
GREEN BLACK BLACK YELLOW RED
a. red
b. green
c. yellow
d. black
4. Which of the following genes would be highly expressing in yeast grown in aerobic conditions:
a. gene to bring sugar into the cell
b. gene to make sugar into alcohol
c. gene to attach carbon to oxygen to make carbon dioxide
d. all these genes would express in all conditions
Post Test
Cells in Space
1. Which of the following describes a DNA microarray:
a. a new technology that allows scientists to view real time gene expression
b. a method of isolating DNA so it can be used for gel electrophoresis
c. the form in which DNA is packaged into chromosomes
d. the graphic results of a forensic DNA sequencing profile
2. DNA arrays can be used for all of the following except:
a. identify whether someone has a certain disease
b. tell whether the genes for living in anaerobic conditions are transcribing
c. genetic ID card
d. predict whether someone will die of heart disease
3. The following diagram displays the results of a particular DNA array that was made through
stander methods: Which color spot(s) indicate that all of the target genes are all transcribing:
O
O
O
O
O
RED GREEN YELLOW BLACK RED
O
O
O
O
O
GREEN BLACK BLACK YELLOW RED
a. red
b. green
c. yellow
d. black
4. Which of the following genes would be highly expressing in yeast grown in anaerobic conditions:
a. gene to bring sugar into the cell
b. gene to make sugar into alcohol
c. gene to attach carbon to oxygen to make carbon dioxide
d. all these genes would express in all conditions
Cells in Space
or “Blast Through That Array, Scotty…….”
In this activity you will use a simulation to investigate the use of a new procedure that connects
molecular biology, cell biology, bioethics and technology. DNA microarrays are powerful tools used
to measure the activity of cells as they transcribe their genes into proteins that control cellular
biochemical reactions and giving cells their unique functions.
Our Story:
Yeast microbes are probably one of the earliest domesticated organisms. People have used yeast for
fermentation and baking throughout history. Archaeologists digging in Egyptian ruins found early
grinding stones and baking chambers for yeasted bread, as well as drawings of 4,000-year-old
bakeries and breweries. Only in the last 150 years, since the experiments of Louis Pasteur, have
scientist begun to explore how yeast works. Pasteur first proposed the production of carbon dioxide
from yeast as responsible for raising a loaf of bread in 1859.
Beer is made by fermenting a plant carbohydrate, usually a starch from a cereal grain such as barley.
The grain seed is germinated in a process called “malting”. During malting the starch is broken down
into small, simple sugars such as glucose. The sugar is then converted into other compounds
including ethyl alcohol. The yeast species of choice is the yeast Saccharomyces cerevisiae.
Yeast cells
All living organisms use respiration to obtain chemical energy from a carbohydrate food source. The
yeasts, like most fungi, respire oxygen (aerobic respiration), but in the absence of air they derive
energy by fermenting sugars and carbohydrates to produce ethanol and carbon dioxide. When yeast
are supplied with both sugar and oxygen, the colonies grow up to 20 times faster through cell division
than without oxygen. In 1815, Guy-Lussac understood how yeasts convert the simplest sugar,
glucose (C6H12O6) to ethanol: C6H12O6 (glucose)2CO2 (carbon dioxide) + 2C2H5OH (ethanol).
Unlike many kinds of fermenting bacteria (such as yogurt making or lactic acid microbes), yeasts
don't require anything but sugar and water to maintain fermentation and growth. For example, their
nutrient broth can be free from other complex molecules such as amino acids, minerals or vitamins,
since the yeasts' history of austere conditions in nature has brought them to a unique state of selfsufficiency, even by microbial standards. The ingeniousness of adaptation makes yeasts one of the
most studied and robust microbes.
A yeast fact: As little as two pounds of yeast starter can raise 500 pounds of bread dough.
Which, of course, makes it a perfect item to bring along on the next space mission. Moon base alpha
has been built and the next launch of the space shuttle will bring the first group of colonists who will
live at the base for a year. Along with the scientists and crew will be a chef who will be responsible
for making their meals. Chef Emeril de Spock has been investigating which yeast strains would be
the best to bring along. He knows the colonists will need to eat bread- and will want to drink root beer.
He doesn’t have enough space to bring different kinds of yeast, so he must bring one that can be
used for both applications- fermentation and baking. Of course, he has a fermenter and an oven on
board. But he can’t decide between several different strains of yeast.
The yeast’s ability to ferment or not is due to the yeast’s genes that transcribe to make specific
proteins and enzymes used in the cell’s respiratory pathways. In real yeast cells this would be due to
the action of thousands of protein products that have to be made at the right time, in the right cellular
place, in the right configuration and in the right amount. Obviously this is a very complicated process
in “real life”. In this scenario, however, we will simplify things and study three sample genes: the one
that allows the yeast to bring sugar into the cell (called S1). Once in the cell, the bonds in sugar are
broken in a process called glycolysis. If the cells are living in a low oxygen environment (anaerobic),
and the second gene of interest is functioning (called F1) then fermentation occurs and the glucose is
converted to carbon dioxide and ethyl alcohol. In other words- needed to make beer. If the cells are
growing in an oxygen rich environment (aerobic), and the third gene is active (called B1) then the
sugar fragments enter the Krebs Cycle and the carbons from the sugar react with the oxygen and
form lots of carbon dioxide. Great if you want nice, light, doughy bread. Obviously the best yeast
would be one where all three genes are active to a great degree.
Using current techniques that you have used in class, such as restriction digests and sequencing, we
can certainly check to see if the genes are there. But, do they really work as intended?
For that, we may use a newer technique called a microarray. A microarray is a tool to analyze gene
expression. It is made from a small membrane or glass slide that contains samples of many genes
arranged in a regular pattern. All our yeast cells contain the same set of chromosomes and genes.
Only a fraction of these genes are transcribed (or turned on or “expressed”) at any particular time.
"Gene expression" is the term used to describe the transcription of the information contained within
the DNA, into messenger RNA (mRNA) molecules that are then translated into the proteins, such as
the enzymes in the yeast, that perform most of the critical functions of cells.
By studying the kinds and amounts of mRNA produced by a cell we can learn which genes are
expressed. This provides insights into how the cell responds to its changing needs- such as low or
high oxygen levels. This mechanism acts as both an "on/off" switch to control which genes are
expressed in a cell as well as a "volume control" that increases or decreases how much expression
(or transcription) of particular genes as necessary.
Two recent complementary advances, one in knowledge and one in technology, greatly influence the
study of gene expression and the discovery of the roles played by specific genes. As a result of the
various genome projects, including the Yeast Genome Project, researchers have identified the DNA
sequences of large numbers of genes. The challenge currently facing scientists is to find a way to
organize and catalog this vast amount of information into a usable form.
The second advance may enable the identification and classification of this DNA sequence
information and identify the functions of these genes. A microarray works because of the principle
that an mRNA molecule will bind specifically to (or hybridize to), the DNA template from which mRNA
originated. By using an array containing many DNA samples, scientists can determine, in a single
experiment, the expression levels of hundreds or thousands of genes within a cell by measuring the
amount of mRNA bound to each site on the array. With the aid of a computer, the amount of mRNA
bound to the spots on the microarray is precisely measured, generating a profile of gene expression
in the cell.
Microarrays are a significant advance both because they may contain a very large number of genes
and because of their small size. Microarrays are therefore useful when one wants to survey a large
number of genes quickly or when the sample to be studied is small. Microarrays may be used to
assay gene expression within a single sample or to compare gene expression in two different cell
types or tissue samples, such as in yeast grown in different oxygen levels or healthy and diseased
tissue. Because a microarray can be used to examine the expression of hundreds or thousands of
genes at once, it promises to revolutionize the way scientists examine gene expression. This
technology is still considered to be in its infancy; therefore, many initial studies using microarrays
have represented simple surveys of gene expression profiles in a variety of cell types. Nevertheless,
these studies represent an important and necessary first step in our understanding and cataloging
genomes.
DNA Microarrays are small, solid supports, usually coated glass microscope slides, onto which the
DNA sequences from thousands of different genes are attached at known, fixed locations. The
American Heritage Dictionary defines "array" as "to place in an orderly arrangement". It is important
that the gene sequences in a microarray are attached to their support in an orderly or fixed way,
because a researcher uses the location of each spot in the array to identify a particular gene
sequence. The spots themselves can be DNA, cDNA (DNA transcribed from mRNA- like a copy or
converted DNA), or oligonucleotides (small strands of single stranded DNA).
One might ask, how does a scientist extract information about different genetic conditions from a
dime-sized glass or silicon chip containing thousands of individual gene sequences? Microarray
technology is based on three fundamental principles of DNA:
 complementary binding of nucleotides: A-T, C-G
 DNA double helix can be separated into single strands (as in the beginning stages of
replication)
 RNA can be changed to single stranded DNA – called cDNA (as in HIV, an RNA virus, making
DNA to infect the cell’s DNA)
Our yeasts will be grown in aerobic and anaerobic conditions. The mRNA will be isolated and
converted into cDNA and attached (or tagged) to a laser-activated fluorescent dye. Although these
dyes are usually red and green, our dyes will be red and blue.
Our fluorescently labeled nucleic acid molecules serve as “mobile probes" to identify complementary
molecules attached to the slide. When two complementary sequences find each other, such as the
immobilized target DNA and the mobile probe cDNA they will lock together, or hybridize.
After this hybridization step is complete, a researcher will place the microarray in a "reader" or
"scanner" that consists of some lasers, a special microscope, and a camera. The fluorescent tags are
excited by the laser, and the microscope and camera work together to create a digital image of the
array. These data are then stored in a computer, and a special program is used to calculate the redto-green fluorescence ratio for each microarray spot by analyzing the digital image of the array. The
program then creates a table that contains the ratios of the intensity of red-to-green fluorescence for
every spot on the array. The computer may conclude that both yeast types express gene S1 at the
same level, that when grown in anaerobic conditions, yeast type Bam1 expresses more of gene F1,
or that in aerobic conditions yeast 2 expresses more of B1. Of course, there could be a gene that
neither expresses! But remember, this is a simple example used to demonstrate key points in
experimental design. Some microarray experiments can contain up to 30,000 target spots. Therefore,
the data generated from a single array can mount up quickly.
To see how a microarray works, go to: http://www.bio.davidson.edu/Courses/genomics/chip/chip.html.
We are going to set up our chip a little differently because … we don’t have real microarrays and
robots to spot them.
Go to http://www.bio.davidson.edu/Courses/genomics/arrays/arrayer.html to see a real, microarrayer
robot in action. We will be doing a mock microarray analysis.
Before you begin the experiment, perform the paper microarray activity. If you can do this AND
understand the computer description, you are ready to test the yeasts:
Test yourself by answering the following:
1. Describe an instance where DNA microarrays use each of the above (a,b,c) principles.
2. Why is the sample RNA changed to cDNA?
3. Why does the DNA have to be tagged with a red or blue dye?
4. What needs to be done to actually see the dye?
5. Can you actually see the DNA molecules on the microarray?
The Activity:
Chef Emeril de Spock has grown several yeast strains in a nice sugary rich broth in both aerobic and
anaerobic environments. He extracted the mRNA from the growing yeast cells, converted it to cDNA
and tagged the samples taken from anaerobic conditions with a blue fluorescent dye and the sample
from aerobic conditions with a red fluorescent dye. He mixed each strain’s cDNA samples in a tube.
Each lab group has a particular strain to test.
Now it is time to test how active the three genes are:
Materials: for each lab team
Samples:
A. reaction tube with your tagged yeast cDNA sample
B. reaction tube for + control, red
C. reaction tube for + control, blue
D. reaction tube with – control (no color)
E. Probe solutions: tubes with single stranded DNA samples with known sequence for S1, F1
and B1. These are in an adhering solution that will harden and attach the probes onto the
microarray slide. They must remain in a waterbath as liquid until use.
Equipment:
 plastic droppers- one for each B, C, D, ES1, EF1, EB1.
 “microarray” slide
 slide developing tray





tweezers
paper towels
colored pencils (red, blue, purple)
waterbath w/ probe solutions
goggles, gloves
Procedure: Although the techniques are not difficult, the materials must be handled carefully because
they are fragile.
1. Place the micropipette slide flat on your lab surface. Label your slide like the diagram. Using your
plastic droppers, place a drop of the following onto one long edge of your slide. Notice that the drops
are aligned along the edge, leaving the opposite edge to handle the slide.
E
S1
E
F1
E
B1
B
C
D
Answer: a. What is the purpose of each of the control drops, “B”, “C” and “D”?
2. Allow the slide to dry. This should take about 5 minutes.
3. Pour the yeast cDNA solution “A” containing the mixture of yeast expressed genes, S1, F1 and B1
into the slide developing try. Using tweezers gently lower the slide, sample side down, into the try.
The opposite side should remain above the liquid. Let stand for about 3-5 minutes.
b. What is occurring at each of the sample spots (“A” – “E”) during this time?
4. Using tweezers gently remove the slide and place flat on a paper towel to air dry. During this time,
the color should develop.
c. There are four possible “colors: red, blue, purple, no “color”. What does each indicate?
d. Color the spots on your array as shown by your results:
E
S1
E
F1
f. What does each of your spot results indicate?
E
B1
B
C
D
g. Compare your results with your class:
S1 _____________
F1: _____________
B1: ____________
h. How should the chef interpret these results?
In this example we only investigated three “genes” and their transcribed products. Obviously yeasts
have many more genes that participate in cellular respiration processes and are differentially
expressed at different times and under different conditions. If this were a real experiment, those
genes would also be sampled and their products probed on the microarray.
Extension:
Read the following article from Time magazine, January 11, 1999 Vol. 153 No. 1 for another
perspective on the use of microarrays.
http://www.time.com/time/archive/preview/0,10987,989981,00.html