Introduction to Genetics Laboratory HNS 123 M or Th 2-4:50 Instr: Gilchrist Office Hours: M 10:00-11:30, others by appointment (arrange by email at gilchrist@ncf.edu or by phone 359-4275) class webpage at www.ncf.edu/gilchrist and webBoard for information and discussion at webboard@ncf.edu/~SandraGilchrist.

Week #             Topic                                                                     Readings and experiments

    1              Safety, Lab Safari, and set up for next week                                           Lab handout 1 and fly manual;

                     Introduction to Drosophila, Fast Plants, chlamydomonas,                 chi squared information

                    and C. elegans;  START QUANTITATIVE FAST PLANT EXPT

     2             Monohybrid Drosophila, Arabidopsis, and C. elegans                          Lab handout 2; mitosis

                    Dihybrid and trihybrid crosses; introduction to probability

    3              Mendelian modifications, sex and probability                                           Lab handout 3; meiosis

                    DISCUSS OPEN EXPERIMENTS

     4            Linkage and crossover; start open ended experiments                             Lab handout 4

    5             Sex determination, sex chromatin, and human cells                                    Lab handout 5, 6

    6-7         Wrap up experiments; clean lab    Hand in reports by Monday of Break week  

     8            BREAK

    9             Intro to DNA technology, lab safety, preparations for E. coli, et al.    Chapters 1-4 and Lab 1,2

                                                                                                                                             Lab handout 7

   10            Restriction analysis                                                                                        Chapters 5,6 and lab 3,4

   11            Colony transformation and resistance; Intro to pVIB; OPEN EXPTS    labs 5,6,8; ; lab handout 8

   12            Purification/Identification of recombinant GFP                                          lab 7; ch 7, 8

   13            PCR Human Alu; Arabidopsis                                                                      lab handout 9

   14            Plasmids and recombination                                                                         lab 9, 10 or 11, 12

   15            wrap up and clean up HAND IN REPORTS/NOTEBOOKS BY MONDAY OF EXAM WEEK

    Links of interest

sdb.bio.purdue.edu/dbcinema/kaufman/kaufman.html this link is to a series of animations on fruit fly development

You will have several pages of information for this experiment. Please make sure that you read through all of the material before you begin your planting and manipulations. We will be working with a Brassica species for part of the experiments in the class. You will have available some plants that have already been started from seeds several days in advance. Pay attention to the types of plants available and consider what you want to find out in your experiments. Careful planning can save a lot of time and effort on your part. There is an extensive booklet online concerning the hairiness selection project at www.mtholyoke.edu/~msrivast/Fast%20Plants%202002.PDF. A copy of the booklet is in a notebook at the end of each bench.

We are going to view the hairiness of Brassica rapa with a twist. Instead of using plain soil in our experiments, we are going to inoculate a part of the soil with mycorrhizal fungi to see if an environmental variable will influence the hairiness of the plants. We will be using an inoculum of ectotrophic mycorrhizae that form a covering of hyphae around the root (for recent senior theses on mycorrhizae see projects of Jessica Palenchar on tomatoes and Sarah Blanton on wetland plants). The Cruciferae, in general, do not have associated mycorrhizae, but the conditions for the genetically altered FastPlant Brassica species have not been described. Decaying Brassica roots release isothicyonates that can function as a fungicide, making them a potential natural biofungicide. If such crops are used as a cover crop between plantings, the mycorrhizal composition of the following crop is generally depressed. However, research from Ryan (2001) suggests that the composition decreases because of the lack of a host crop rather than from the decomposition products. Some have indicated that certain strains of Brassica do form associations with mycorrhizae, though these experiments have not been repeated and are called into question. You will find in your lab handouts a copy of "Importance of Mycorrhizae for Agricultural Crops" by Muchovej (1991) published as SS-AGR-170 from the Institute of Food and Agricultural Sciences at University of Florida.

You will be required to make a map of the lab and surrounding area for your notebook. Be sure to label major landmarks in the lab. Locate the following in the lab and make sure that they are noted on your map.

Eye wash station

incubator

autoclave

red biohazard disposal

trash can

sharps container

petri dishes (note drawer numbers)

flasks

deionized water

goggle cabinet

safety shower

greenhouse

dishwasher

microscope slides (note drawer numbers)

scissors

drawer indices

paper towels

Once you have your map drawn, compare the results with your lab partner.

Probability and genetics with a hint of statistics

 Mendel’s work has as its underpinnings ideas of probability.  He is often credited with linking mathematics to heredity to understand how traits are passed to future generations. (However, camel breeders in Egypt and silkworm breeders in China were using such calculations long before Mendel was born!)   Random events such as mating allow use of some powerful forms of mathematics in predicting outcomes.  It should not be surprising that some of the major tenets of Mendelian genetics are stated in terms of these probabilities, making genetics one of the few scientific endeavors to have such a basis.  In exploring ideas of both genetics and probability, you will learn to think of independent events that can occur simultaneously and of events that are mutually exclusive.  You will also be asked to draw upon ideas from algebra and calculus concerning binomial expansion to examine combinations of events.  A little terminology is needed to really gain a good understanding of what you need to know.  First, mutually exclusive events are those that have no common points; they cannot occur at the same time.  This can be expressed as P (A and B)=0.  Independent events occur without relation to each other.  A conditional probability gives the likelihood of an event occurring providing that a second event has already occurred (probability of B given A).

 You are familiar from other classes with the idea of “chance”.  You hear the term commonly in language every day.  In genetics, the ideas surrounding chance have very important applications.  Perhaps the simplest activity that you can recall involving chance in classes is when you tossed a coin to determine if the chances of having a head or a tail land face up have equal probabilities.  Though this may seem irrelevant to genetics, there are many instances when there is a 50:50 chance that an event will occur.  Take a coin and flip it 50 times.  Record the number of heads and the number of tails.  If the coin is weighted fairly, do you expect an equal number of heads and tails?  If so, then your expected for the event is 25 (50%).  So, subtract the expected from the observed to get the deviation.  If you flip the coin more, do you think that the number will more closely approximate 50:50?  Why?  Try it.  Flip the coin 50 times more and add the results.  Now look that the deviation.  Is it smaller?  What does this tell you about the coin?  What happens if you have a deviation that is relatively large?  Can you attribute this to chance alone or must you examine for another factor?

 Now, examine two events.  Take two coins and flip them simultaneously for 50 times.  Record your results as before.  The probability of two or more independent events happening simultaneously is the product of their individual probabilities.  Therefore, if you are looking at the probability of a coin landing with the head facing up, it is ½ and it is the same for tails.  The probability that both will be heads is ½ x 1/2= ¼.  How would you calculate the probability for having a head and a tail face up.  The first part is easy, the probability that the first coin is a head is ½ and the probability that the second coin is a tail is ½.  However, there is a second way that the combination can be achieved.  If the first coin is a tail and the second is a head, then the outcome is the same as the first.  So, 2 (1/2 x ½) =1/2 .  What do you think will happen if you flip the coins more times.  What would be the expected if you were flipping three coins?  Can you think of a genetic situation where knowing the probability of three independent events would be useful (Hint:  think about children in a family)?  Remember that if you are looking at isolated events, it is difficult to predict the outcome.  What you are calculating is a probability from a total number of events.  Therefore, if you want to know the probability of having 4 girls in a family it is an easy calculation.  However, if you want to counsel a specific family on the probability, you have to indicate many factors that can affect the outcome within a specific unit.

 The conditional probability is very powerful in genetics, especially in examining pedigrees.  We can apply Bayes’ theorem that states, P (B|A)= P(A and B)/P (A).

 Statistical tests

 One of the most useful basic statistical tests in genetics is the chi-squared test.  The many forms of the test are used in various ways.  The test is specifically designed for data in the form of nominal categories.  Before one can apply this type of test, some basic tenets must be met.  First, the samples must be independent.  This goes back to the notion of the conditional probability.  One observation in the sample set must not affect another observation; otherwise, this test is violated and the outcome of the calculations are suspect.  One of the most common errors in terms of independence is what is referred to as serial correlation.  This is a time dependent relation.  For example, if one is sampling plankton on an incoming tide, starting from offshore and moving towards shore, then depending on the interval it is possible to be sampling the same water just displaced shoreward rather than sampling a different area of water.  Sometimes, you might encounter outliers in data.  Outliers can be a result of poor data recording (transposing numbers, for instance, decimal point in the wrong place, misreading latitude and longitude on GPS, etc) or may be the result of sampling a mixture of populations.  This sometimes occurs in looking at gendered variations such as height.  For example, within a sample of females, one may have close to a normal distribution of weights.  Within a group of males, one may also have a normal distribution of weights.  Combining the two distributions may not give a distribution where the mean, median and mode are equal (definition of a normal distribution).  It is likely that there will be some skewing in the data away from the mean and there will likely be some effects on the high and the low end of the combined populations compared to the individual groups (often referred to as the kurtosis of the distribution).  The data are typically in the form of frequencies.  Percentages are not used.  Typically, though not absolutely required, the minimum number of observations in a category is 5. It is important for you to know how to handle zero values in this type of statistic.  There are two basic types of zeroes.  The first is called a sampling zero.  This is a zero value for a cell and is acceptable as long as such values do not constitute a major portion of the total cells.  A structural zero is not acceptable and will violate the underlying assumptions of a chi-squared test.  A structural zero is a cell that can never have a value other than zero.  This constitutes a design flaw in the test. Though the data are generally categorical, one can use continuous data carefully.  The data must be divided into reasonable, defensible intervals.  This means that one must determine reasonable intervals before the data are collected.  For instance, if one is looking at fruit fly bristles, a continuous variable would be count.  One could set an interval of less than 5, 6-10, 11-20 and over 20 per square millimeter to indicate a level of hairiness, for example.  Overall from this statistic, we are trying to determine if the observed values and expected values differ by chance alone.  Thus, the null hypothesis is that there is no difference between the observed and the expected values.

 One of the most difficult concepts to understand is the notion of degrees of freedom.  Generally, degrees of freedom represent the number of scores that can change without altering the underlying distribution of values.  For a chi squared test, the degrees of freedom determine the height, center and skewness of the distribution.  You will need to look at a table of chi squared values, determine the degrees of freedom that you have, and the level of error you will accept—commonly 5%.  To help with understanding the goodness of fit chi squared test, you might want to look at  www.georgetown.edu/faculty/ballc/webtools/web_chi_tut.html  or davidmlane.com/hyperstat/index.html for assistance with understanding how to apply this enumeration statistic to your results.

Mono and Dihybrid crosses for Genetics class

You should be familiar now with the general anatomy of the Drosophila, especially the mutants that you are culturing and the wild types that you have examined.  You will take note of the offspring and consider whether the ratios formed are those expected from Mendelian predictions.

 Monohybrid cross

You should have a single mutant (chromosome 1, 2, 3 or 4) culture.  You will need to create an F1 and record their phenotypes.  Then you will need to cross the F1 generation to create an F2; this can be done when you have enough males and females for mating.  The data for the F2 should also be recorded.  You should begin recording information and separating flies as soon as they start to emerge.  Generally, higher numbers of females emerge first.  Count the flies every other day for at least 10 days for both the F1 and the F2 so that you will get a reasonable representation of the offspring.

 Last week, you set up a vial of the mutant parents and the wild types.  So, you can remove the parentals from each of these. This will allow you to collect virgin flies without a problem of confusing the parentals with the newly emerged flies.  The flies that emerge constitute the F1 generation.  Collect at least 3 females and 2 males and create your F2.  So, you will have an F1 vial for the wild type and for the mutant.  The F1 for the wild type will allow you to evaluate emergence rates and sex ratios.

 Dihybrid cross

Use your mutant stock of the 1-4 chromosome flies to mate.  Cross a 1-4 chromosome mutant with a mutant on a different chromosome.  For instance, if you have a chromosome 1 mutant, cross it with a chromosome 2, 3 or 4 mutant.  Carry this through to the F2, counting the offspring. 

Experiment on alcohol tolerance using the fruit fly Drosophila melanogaster

(I strongly  suggest that two groups combine to do this set of experiments)

 Fruit flies are found commonly in areas where fermentation is occurring.  They are attracted to fruit fermentation sites for oviposition.  Larvae feed on fruit as they mature to adults.  The flies possess a gene called Adh that controls production of alcohol dehydrogenase that gives this the ability to oxidize ethanol to NADH and a aldehyde (or ketone) and H⁺.  Mutant flies that are Adh- cannot process ethanol and show very obvious behavioral differences from their wild type conspecifics.  The mutant flies have difficulties flying, walking, and engaging in social interactions.  If too much ethanol is consumed, the flies will succumb to alcohol poisoning.  In humans, Adh concentrated in the liver helps to breakdown ethanol.  However, women tend to have less Adh associated with liver tissue and they tend to have a different ratio of fat to water content in the body.  There is a suggested trend that women who abuse alcohol acquire liver and brain damage more quickly than their male counterparts, even after adjusting for differences in metabolism and weight.  Alcohol equilibrates with water content of about 68% for men and 55% for women (women have less water and more fat in their body tissues, in general, than men).  The alveolar air reflects this equilibration, forming the basis for a breathalyzer test in humans.  For the flies, the amount of body fat and water content difference is negligible.

Experiment 1

 For this experiment, you will set up two different types of vials.  One will be without substrate and will be for observing behaviors only.  The second type will be with the blue Drosophila substrate.  For the first set of vials, you and your partner(s) will isolate 15 wild type and 15 Adh mutant flies (try to get an even mix of sexes, though not required).  Place a cotton ball into each of the observation vials.  To two vials each, add 5 mls of water.  Make sure that the liquid is absorbed by the cotton ball; pour off excess and dry the insides of the vials.  Now, in second set add 5mls of beer and repeat.  To third set, add 5 mls wine and repeat procedure.  You should have a total of 6 vials.  Add 5 wild type flies each to a water, wine and beer vial.  Add 5 Adh mutant flies each to a water, wine and beer vial.  Lay the vials on their sides until the flies are fully awake so that they do not drown in the wet cotton.  Note the behavior of the flies at time zero then at 30 minute intervals for the next two hours.  Subsequently, note the behavior of the flies after 8, 16 and 24 hours exposure.  Compare your observations with others in the class.

 Experiment 2

 For the second set of vials, you are going to use the water, beer and wine to moisten the substrate.  You will have to dilute the wine and the beer for the experiment to 2% solutions.  Add about two centimeters of substrate to 6 vials, moisten two with water, two with dilute wine and two with dilute beer.  Then add at least three female and two male wild types to each type of vial and the same number and sexes of the Adh mutant flies to the remaining vials.  Allow the flies to mate and observe the number of larvae that emerge and the pupae that form.  Note any differences that you see in the vials. 

 Experiment 3

 For a final experiment, collect virgin wild type females and Adh mutant females.  Mate the wild type females with Adh mutant males and mate the Adh females with wild type males.   When the heterozygotes emerge,  repeat experiments 1 and 2 using wild type flies for one set of vials and the heterozygotes in place of the Adh flies for the other set in both experiments.  Are the results different for the homozygous recessive mutants and the heterozygous flies?  Explain

Mitosis of plants

For this lab, we will be using a variety of root tips.  We choose root tips for several reasons:  they are easy to obtain, several tips can be harvested from a single specimen, and they can be observed easily.  You will be able to choose from onion or one of the other roots available in the lab.  If you would like, take samples from more than one root type but be sure to label them clearly.

First, use a razor blade to cut the root tip.  The root is the first embryonic organ to emerge from a developing angiosperm. Where do you think that cells are added to the tip, near the plant or at the end?  Take a look at the root and notice that the end has a relatively hard covering.  This is called to root cap and is used by the plant to help push the growing root through the soil.  The cells near the cap form the apical meristem, a fast-dividing part of the root.  Thus, you will want to take off the small amount of material at the tip that is the cap and examine the material immediately above the tip.  To do this, cut off the cap and take about a centimeter of the root to examine.

To make the stages of mitosis more observable, we will stain the root.  However, it is difficult to get stain into the cell because of the cell wall.  So, the first thing that you need to do is to soften the cell wall with warm 1M HCl.  You will find the vials of prepared HCl in the water bath.  BE CAREFUL.  THIS IS AN ACID.  USE GLOVES AND GLASSES.  Place the tips for use into the vial for your group and leave in the water bath for 10 minutes.  What is happening?  The acid is being used to hydrolyze the pectin in the root to separate them.  You are also preparing the DNA for staining by hydrolyzing it.

You will have access to two types of roots:  water only and grown in water plus PDB (p-dichlorobenzene).  PDB is often found in commercial mothballs.

After 10 minutes, carefully pour off the acid from your vial into a waste container on your bench.  The root should feel soft to the touch when you prod it with a dissecting needle.   Blot the tip on a paper towel gently and place onto a microscope slide.  Use a razor blade to chop the root into small pieces.  This also helps the stain to penetrate.  The metal in the blade will react with the stain to give a richer color to the cells, so chop finely and take your time.  Using a GLASS coverslip, cover the tip and fold over the paper towel across the slide.  Add a small drop of acetocarmine to one side of the slip and wick it across using a kimwipe. Tap gently with the eraser end of a pencil or with the wooden end of a dissecting needle.  Place on a warming table for a few minutes.  Look at the cells using a compound microscope.  Can you see different mitotic phases?  What differences do you see in the tips that were water only and those that were grown in PDB?  Can you determine the number of chromosomes in the cells?  Draw and label the different mitotic stages that you observe.  Be sure to label your slides with magnifications and organisms indicated.

Clean off cover slips into the waste containers and rinse slides.  Place them on paper towels to dry.

Mendelian Modifications and Sex:  A tale of three organisms

 We are going to work with three different organisms to examine variations of Mendelian ratios and to explore concepts of sex.  You have already worked with fruit flies and are familiar with their life cycle.  The other two organisms, Caenorhabditis and Chlamydomonas, are commonly used in genetics research.

The diploid organism, Caenorhabditis elegans (or C. elegans), has become an important model organism in genetics because of the ease of tracing cell line development, ability to create obvious somatic and gametic non-lethal mutations, variability in sex with a hermaphroditic form and a male form (typically smaller than hermaphrodites and with broader tail), and the ease of keeping the animals in the lab.  Another very valuable feature is that the covering of the animal is relatively transparent, revealing inner structures clearly. For the invertebrate zoologist, the nematodes are an interesting representative of the pseudocoleomate body form.  The outer covering of the animal is a tough cuticle that is flexible, allowing the animal to have relatively high hydrostatic pressure inside.  The cuticle must be shed for the animal to grow, so during the maturation period, the larvae will typically go through 4 molts.  Like other nematodes, the animal moves by “lashing” as a result of four longitudinal bands of muscles.  Nearly 17, 800 genes have been identified in the organism, including both nuclear and mitochondrial genes.  Today, both genetic structures have been sequenced.  They have a relatively simple natural history.  Unlike many of the nematodes, they are free-living. They feed off a common lab bacterium, Escherichia coli (E. coli).  In the field, they consume a variety of bacteria found in the soil.  The reproductive system of the hermaphrodite is very interesting.  It consists of uteri, oviducts, spermathecae, and ovaries.  These connect at the uteri and empty to the outside via a structure called a vulva.  Keep in mind that the hydrostatic pressure inside of the worm is high relative to surroundings, so extending the vulva is not difficult.  Holding it in place takes energy.  Depending on the species of Caenorhabditis, the vulvar opening can be simple or ornate.  Sperm are made before eggs and are stored until needed.  The structure of the male form is somewhat different.  A cloaca is formed by the male gonad and the vas deferens.  Expulsion of the sperm is via an elaborate copulatory bursa.  White, et al. 1986 wrote an elegant paper in the Philosophical Transactions of the Royal Society of London, Series B, Biological Science Vol.314, Issue 1165, pp 1-340 entitled, “The structure of the nervous system of the nematode Caenorhabditis elegans that forms a basis for much of our understanding of the basic biology and function of the nematode.

What you will do:  BE SURE TO SIT DOWN AND PLAN OUT YOUR TIME AND WHAT YOU ARE GOING TO DO.  MUCH TIME AND EFFORT WILL BE WASTED IF YOU TRY TO DO THIS LAB WITHOUT PROPER PREPARATION!

The first thing that you will do is to make your own culture of C. elegans.  One plate will be available for you already, so you will need to make 5 more.  To do this, you will need to make nutrient agar.  HOWEVER, BEFORE YOU DO ANYTHING YOU WILL NEED TO STERILIZE YOUR WORKPLACE.  USE LYSOL TO CLEAN THE SURFACE OF THE COUNTER.  KEEP OBJECTS, INCLUDING YOUR ELBOWS, OFF THE SURFACE AFTER IT IS CLEANED.

Making nutrient agar is simple; you are going to have at least 6 plates per group.  Follow the recipe on the container.  Autoclave the agar and the plates for 10 minutes.  You will need to pour the agar into sterile plates and allow the media to cool and set. 

You will find a culture of E. coli already in the lab for you use in the incubator. Be sure that you are wearing gloves and eye protection.  You need to pour about 10 mls of the material into a sterile container.  You will find sterile containers on each of the benches.  Now, use a sterile pipette and place about 1 ml on the surface of your agar plate and use the spreader to disperse the material over the surface.  Allow the surface to dry for at least 30 minutes in the incubator. After this, your plate is ready to accept the C. elegans.  Once you have your plate poured and ready, sterilize a scalpel, allow it to cool down, carefully open the lid of the stock culture and excise a block of agar.  Place the block with the culture side touching the surface of your new plate.  You can incubate the worms at 20^oC for optimal growth.

You are now ready to self the culture.  Isolate a single hermaphrodite and place it onto an agar plate.  Males will occur spontaneously in the hermaphrodite progeny at a relatively low frequency, so you will have to be on the lookout for males.  Once located, you can transfer a few males to a plate with a few hermaphrodites to increase the number of males in your culture and to maintain a stock of males.

As you can see from the attached figure, on day 1 the adults will lay eggs on the surface of your culture plate.  By day 2, some of the eggs will have embryos that have developed fully and are hatching.  There may be some larvae on the plates.  Day 3 you will see larger worms as the larvae continue to mature.  Typically, after the fourth molt, the reproductive organs are fully formed and are clearly visible.  These animals are now fully reproductive.

You can alter the growth temperature of the worms to examine the effects of temperature on reproduction.  Observe the number of males produced under each condition.  Does it change?  Do you see other morphological changes?  Plate some of the worms from the lower temperature and some from the higher temperature onto new agar.  Place one set into the same temperature as originally grown and switch another plate to the different temperature.  Do you see a change in the reproduction?  What is happening genetically?  How would you test your hypothesis?

A select set of references that might be of interest in learning about C. elegans

Abad, P., Quiles, C., Tares, S., Piotte, C., CastagnoneSereno, P.,  Abadon, M., and Dalmasso, A. 1991. Sequences homologous to Tc(s) transposable elements of Caenorhabditis elegans are widely distributed in the phylum nematoda. J. Molecular Evolution. 33: 251-258.

 Ahnn, J. and Fire, A. 1994. A screen for genetic loci required for body-wall muscle development during embryogenesis in Caenorhabditis elegans. Genetics 137: 483-498.

 Ahringer, J. and Kimble, J. 1991. Control of the sperm-oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 3 untranslated region. Nature 349: 346-348

 Bargmann, C. 1993. Genetic and cellular analysis of behavior in C. elegans. Annual Review of  Neuroscience 16: 47-71.

 Beitel, G., Clark, S., and Horvitz, H. 1990. Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348: 503-509

 Driscoll, M and Gerstbrein, B.  2003. Dying for a cause: invertebrate genetics takes on human neurodegeneration.
Natural Review of Genetics 2003 Mar 4(3):181-94

 Fatt, H. and Dougherty, E. 1963. Genetic control of differential heat tolerance in two strains of the nematode Caenorhabditis elegans. Science 141: 266-267

 Salser, S. and Kenyon, C. 1996. A C. elegans Hox gene switches ON, OFF, ON, and OFF again to regulate proliferation, differentiation, and morphogenesis. Development 122: 1651-1661

Strange, K.  2003.  From genes to integrative physiology: ion channel and transporter biology in Caenorhabditis elegans. Physiological Review  Apr 83(2):377-415.

 Walthall, W. and Plunkett, J. 1995. Genetic transformation of the synaptic pattern of a motoneuron class in Caenorhabditis elegans. Journal of. Neuroscience: 1035-1043.

 White, J.G., Southgate, E., Thomson, JN and Brenner, S.  1986. The structure of the nervous system of the nematode Caenorhabditis elegans.  Philosophical Transactions of the Royal Society of London, Series B, Biological Science Vol.314, Issue 1165:  1-340.

Chlamydomonas

Chlamydomonas is a very interesting unicellar alga belonging to the Chlorophyta. Most recently, researchers have become interested in how the alga senses light and responds to it using a rhodopsin-like protein.  Though there are more than 500 known species that inhabit terrestrial and aquatic areas, we will work with one of the most popular species for study in genetics.  It is a generally a motile organism.  An advantage of using this organism is that the generation time is about 5 hours!

 What we will do:

 You will be mating Chlamydomonas and observing the behavior of the organisms.  Prior to lab, the instructor will activate the slant cultures by placing them under a light for two hours.  You will add mating solution to the cultures and replace them under the lamp. After one hour, remove a small amount of the culture onto a slide and observe the organisms. Watch how they move and interact.  After 2 hours under the lights, you can begin the mating experiments with the cultures.  Invert the tubes gently to shake the contents.  Using a sterile pipette, take a drop of minus culture and place it onto a slide. Add a drop of plus strain using a different pipette.  Quickly place the slide under the microscope and observe.  Mating will happen quickly as you can note by the clumping of the cells.  You will start to see mated pairs move from the clump.  Plasmogamy (fusion) will begin to occur within a half an hour.  The zygospore will swim for a short time, but will eventually settle to the bottom after losing its flagellae.  Once the flagellae are lost, the organism is called a zygote.  You can try some activities to observe variations in the organisms.  Select those that seem most sensitive to light, etc.

Deininger, W., Fuhrmann, M. and Hegemann, P. 2000. Opsin evolution: out of the wild green yonder?  Trends in Genetics 16, 158-159

DiBella, L. and King, S.  2001. Dynein motors of the Chlamydomonas flagellum. International Review of Cytology 210, 227-268.

Ferris, P.J., Armbrust, E.V., Goodenough, U.W. (2002) Genetic structure of the mating-type locus of Chlamydomonas reinhardtii. Genetics 160: 181-200.

 Ferris, P.J., Pavlovic, C., Fabry, S., Goodenough, U.W. (1997) Rapid evolution of sex-related genes in Chlamydomonas. Procedings of the Naional Academy of  Sciences USA 94: 8634-8639.

Losi, A., Kottke, T., and Hegemann, P. 2004.  Recording of blue light-induced energy and volume changes within the wild-type and mutated Phot-LOV1 domain from Chlamydomonas reinhardtii.  Biophysical Journal 86, 1051-1060

Linkage and crossover (expts for week 4)

I URGE YOU TO MAKE A CHART OF WHAT YOU ARE GOING TO DO IN THIS EXERCISE SO THAT YOU WILL HAVE APPROPRIATE FLIES READY FOR MATING!

 Mendel was indeed a clever natural historian and mathematician.  He “discovered” some of the basic tenets of transmission genetics.  He clarified the notion that each parent in a sexually reproducing organism contributes material to the offspring and that material is in the form of a distinct unit.  We know that the units are genes and that parents pass along different alleles of a gene to offspring.  The first law of Mendel, the law of segregation, still holds for sexually reproducing organisms.  However, the second law, independent assortment is only true for a narrow set of circumstances.  Mendel had no concept of the chromosome, thus no understanding of how genes might be associated.  Carl Correns in the 1900’s was the first to establish the linkage phenomenon clearly in plants.  We now know that alleles of genes found close together on the same chromosome (syntenic) tend to be inherited together more often than expected by chance alone.  However, syntenic genes may also assort independently if they are far apart.  Proximity can make a difference in how genes interact.

 For this experiment, you will need to mate females of your 1) multi mutant strain (with at least one chromosome 2 or 3 mutation and 2) females of a chromosome 2 or 3 mutant (you should avoid using dumpy because it is so close to the curly locus; see page 23 of Carolina Drosophila Manual) with flies that are curly plum/dichaete stubble.  Curly, Plum (dominant form of brown locus), Dichaete and Stubble are all dominant and are lethal in homozygous forms.  You are going to reveal the linkages by examining patterns in offspring.  Recall from genetics class that if a recessive mutation is on the sex chromosome, it can be revealed in a sex-linked pattern in insects.  For Drosophila, the sex chromosome is designated as chromosome 1.  It is easy in these organisms that have only 4 chromosomes to examine sex linkage.  However, though there are only three autosomes, you will get a hint as to how difficult it becomes to use Mendelian crosses to determine relationships of markers on these chromosomes.  Curly and Plum both occur on chromosome 2 but are non-allelic (they are not the same gene).  Also, these Dominant mutations act as recessive lethals that are maintained in a balanced (repulsed) configuration (see below):

 Cy     +/+     Pm

 This means that curly and plum will not be found on the same homolog in a single individual; so the configuration is maintained from generation to generation.  This same type of configuration holds true for stubble and dichaete that are found on chromosome 3.

 Diagram some of the potential outcomes of crosses.  This will give you an idea of what you should expect to find.  How do the potential multi mutation offspring differ from the single mutation outcomes?  Are any of the outcomes sex linked?  What can you conclude if all of the F1 are dominant?  What would happen if the mutation is recessive?

 If you determine that the mutation is dominant, mate wild type females with F1 males.  Hint:  you should have 4 phenotypes.  Select the phenotype of male that have a mutation that appears farthest from the mutation.  For instance, if you have vestigial, then Cy/D might be a good candidate for the males.  If you have a sex-linked trait, then the mutation will show up only in the female offspring of the F2.  If the mutation is autosomal recessive, what are your expected results?  For example, do you expect to see a chromosome 2 recessive with a curly or plum mutation?

 References of interest

Gudbjartsson, D., Jonasson, K., Frigge, M. and Kong, A. 2000.   Allegro, a new computer program for multipoint linkage analysis.   Nature Genetics 25:12-13

Mester, D.,Ronin, Y., Minkov, D., Nevo, E. and Korol, A.  2003. Constructing Large-Scale Genetic Maps Using an Evolutionary Strategy Algorithm Genetics 165: 2269-2282.

Piccolboni, A. and Gusfield, D.  2003. On the complexity of fundamental computational problems in pedigree analysis.   Journal of Computational Biology 10(5):763-773.

Rubin, G. and Lewis E.  2000. A Brief History of Drosophila's Contributions to Genome Research. Science 287: 2216-2218

Simons, K., Gehlhar, S., Maan, S. and Kianian, S.  2003. Detailed Mapping of the Species Cytoplasm-Specific (scs) Gene in Durum Wheat.  Genetics 165: 2129-2136

Yap, I., Schneider, D., Kleinberg, J., Matthews, D., Cartinhour, S., and McCouch, S.R. 2003. A Graph-Theoretic Approach to Comparing and Integrating Genetic, Physical and Sequence-Based Maps Genetics  165: 2235-2247

Sex determination:  forensic clues

 In humans, recall that females typically have two X chromosomes.  A theory called dosage compensation (Mary Lyon in1961 offered a notion referred to as the “Lyon Hypothesis”), in conjunction with the observation of Barr bodies (described by Murray Barr in 1949) in female cells, indicates that the second X chromosome becomes inactive.  A typical test done in biology labs is called a buccal scrape.  For this class, we will sample the squamous epithelial cells of the mouth (taken from inside of the cheek) and examine these cells for presence or absence of Barr bodies.  Typically, females (in about 10-20% of cells examined; the Barr bodies are observable in interphase cells and their presence may also be obscured by the position of the cells on the slides) will have one Barr body and males will have none. (How many Barr bodies does a person with Turner syndrome possess?) There are exceptions to this general observation if one of the androgen insensitivity syndromes (XY male genotype, female phenotype) or one of the andrenogenital syndromes (XX female genotype, male phenotype) occurs.  Of course, we also know now that the SRY gene region of the Y chromosome can crossover to an X, resulting in an XX^SRY which can show either a male or female phenotype depending on which cells have the X^SRY as a Barr body and the expression in the embryonic development.

 It is critical in this exercise that you rinse your mouth thoroughly.  Debris or bacterial contamination can cause problems with interpreting results.  So, get some water from material provided and swish the water in your mouth for about 30 seconds.  Discard the water.  Rinse a second and a third time in this same manner.  Immediately after the final rinse, use the toothpicks provided to collect cheek cells from the inside of your cheeks.  Each partner will make one slide of his or her right cheek cells and one of the left by placing the scrapings onto glass slides.  You should make two or three scrapes of each cheeks to make sure that you have a large sample of cells as some will not be in the correct mitotic phase, some will be broken or damaged, and some will be folded.  CAUTION:  Any body fluids such as saliva should be treated as potentially hazardous material so wear gloves and glasses and dispose of materials in the biohazard containers Be sure to label the slides with your name, date and right or left cheek.  Now, you are ready to fix and stain the cells.  Place the slides in the fixative for at least an hour.  Be sure to use forceps in handling the slides as any epithelials that you have on your gloves can contaminate your sample.  After an hour, remove the slides from the fixative and place into the containers covered with a paper towel to air dry.  Be sure to keep the container covered by the paper towel to reduce opportunities for contamination by debris.  After about 15 minutes, the slides should be dry (up to 30 minutes may give a better preparation).  Remove the slides from the drying container and place them into the stain for 5 minutes.  IT IS CRITICAL THAT YOU NOT OVERSTAIN THE SLIDES, SO YOU NEED TO TIME THIS STEP.  At the end of 5 minutes, you are going to rinse the slides in 95% ethanol three separate times.  This should loosen and remove excess stains to reveal the nuclear material of the cells.  You will also do three final rinses (1-2 minutes each rinse) in xylol to further dry and fix the stain in the nuclei.  These rinses MUST BE DONE UNDER THE HOOD AS XYLOL IS VOLATILE AND DANGEROUS.  SO, KEEP THE RINSE CONTAINERS COVERED WHEN NOT IN USE.  Allow the slides to dry in the final drying container for at least an hour before observing.  This is a quick peek to see how the slides look (are they too dark, too light, etc).  Return the slides to the drying container and allow them to dry at least over night in the hood.  After this time, you can score the slides by observing them under at least 400X magnification.  You should try to count at least 100 cells on each slide.  Record how many have Barr bodies.  If less than 2% of the cells on a slide have Barr bodies, this is considered a negative result (male genotype) while a percentage of cells higher than 10% indicates a positive (female genotype).  Don’t panic if you get some unexpected results, there are false positives and false negatives due to sampling errors.  Record your results in your notebooks.  What are your conclusions?