FILE S1. Lab 4. One fly, two fly, red fly, white fly. (Okay, so I m no Dr. Seuss): Recombination mapping of Transposon Insertions in Drosophila

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1 2 SI FILE S1 Lab 4. One fly, two fly, red fly, white fly (Okay, so I m no Dr. Seuss): Recombination mapping of Transposon Insertions in Drosophila Introduction In a previous laboratory exercise, we studied the biosynthetic pathway that underlies eye pigmentation in fruit flies (Drosophila melanogaster). Geneticists have taken advantage of our knowledge of the eye pigmentation pathway to create tools that they can use to study and understand other biological processes that occur in fruit flies. One locus that has been used for this purpose is the white locus, a locus that has a gene product that is involved in transporting pigment precursors from the hemolymph (blood) of the fruit fly into the cells that will make up the eyes of the animal. Without this gene product, the pigment precursors cannot be brought into the eye cells, and the cells will appear white. The DNA of the white gene has been identified, isolated, and sequenced. Geneticists have used the white gene as a traceable marker in recombinant DNA constructs. One way that they have been able to use the white locus to do this is described below. Transposons are genetic elements that are capable of moving from one place to another within the genome of an organism. Most organisms from bacteria to humans have transposons, and many organisms have many copies of particular transposons present in their genomes. It is estimated that humans have between half a million and a million copies of the Alu transposon per haploid genome (so multiply by 2 for the number of copies in the diploid genome). Most transposons carry inside of them a gene that encodes an enzyme called a transposase that catalyzes the excision and reinsertion of the transposon in the genome. However, some transposons that lack the transposase gene use the transposase enzyme produced by other transposons to move around. However, under conditions where there is no external source of transposase, these transposons, which can t produce their own transposase, are fixed into place and are genetically stable. By the careful addition and removal of transposase, it is possible for geneticists to control exactly when transposition can take place. P-element transposons were among the first Drosophila transposons to be discovered, and are among the best understood. Each end of a P-element is defined by an inverted repeat. The sequences at either end of a P-element are mirror images of one another (for example: if the bases at one end are CAT, the bases at the other end will be TAC). In a typical P-element, a gene encoding P-element transposase (each type of transposon has a specific transposase), that allows the P-element to move (transpose) from one location in the genome to another. Geneticists have taken these typical P-elements and modified them by removing the transposase gene, and replacing them with a wild-type copy of the white gene. These P-elements are no longer able to catalyze their own movement from place to place in the genome, and require an external source of transposase in order to move. However, unlike the original wild P-elements that are

2 3 SI phenotypically invisible (unless they are interrupting a gene), the P-elements carrying the wild-type white gene can be tracked by eye color. You will remember that wild-type fruit flies are deep red in color. Homozygous or hemizygous mutants at the white locus have white eyes. Putting a P-element carrying a wild-type copy of the white gene into a wild-type fly will not noticeably change the eye color, but putting the same P-element into a mutant white fly will produce a fly with eyes that will be somewhere between light orange and deep orange, but will almost always be lighter than wild-type. This is because the geneticist did not place the whole white locus into the P- element, but instead inserted a mini-version of the white locus that is missing some of the regulatory elements that are found in the native gene. Fruit fly geneticists have used P-elements carrying the coding sequence for the white gene to place copies of the white gene all over the Drosophila genome. Sometimes the P-element will land in a location that does not interfere with the function of any of the genes normally found in fruit flies. Other times, the P-element lands in a location that interferes with the function of a fruit fly gene. These are called transposon insertion mutants. There are a number of ways that a transposon can interfere with the function of a gene, but the easiest one to understand is when the transposon lands within the coding sequence of the gene. This can interfere with transcription and prevent the production of gene product. Many of the interrupted genes have been molecularly characterized by sequencing the DNA on either side of the transposon insertion, and but very few have been mapped genetically. In this lab, we, as a class will be recombination mapping these insertion mutants for the very first time by tracing the red eye color phenotype carried by the transposon in a series of genetic crosses. The transposon insertions that created these mutations occurred in the presence of transposase enzyme, but that enzyme has been removed from the lines and from our mapping strain, so the transposons will not move during our crosses.

3 4 SI Overview of the Next several weeks This week: Setting up the P0 Cross. Lab 7: Bioinformatics lab-finding out what there is to know about your mutant on the web Lab 8: Setting up F1 backcross (Test cross). Lab 10: Scoring the backcross.

4 5 SI Overall Crossing Scheme Transposon line X white mutant mapping line Red eyed F1 progeny X white mutant mapping line (Test cross) Score backcross progeny Materials Drosophila, wild type and white mutants (for comparison with lines carrying P-elements) A unique Drosophila transposon insertion line for each student (acquired from the National Drosophila Stock Center at Indiana University in Bloomington, Indiana) A Drosophila stock that is mutant for the white locus and also carries several phenotypic markers that can be used to recombination map the transposon insert. Fly food Dissecting Microscopes Microscope Illuminator Etherizer and Re-etherizer Metal spatula and/or paintbrush Procedure 1. Your instructor will provide you with a unique stock of Drosophila carrying a transposon insert. Make sure to record the stock number both in your journal for the day, and on the master list for the class, and write your name on the mutant stock bottles you are using. You will be working with the same stock for the next several weeks, so be sure to keep track of your stock number. 2. Transfer a few flies (a whole vial is too many!) into your etherizer, and then examine these flies under the microscope. Make all of the observations in steps 3 and 4, and practice sexing in step 5, always using the same flies, re-etherizing as often as necessary. You will NOT be using these flies in your crosses, only for observations. 3. Make note of the eye color of your strain and record in your journal. If necessary, compare the flies of your strain with Wild-type and white mutant flies. You may detect several eye colors mixed together in your stock vials this is because the white gene carried by the P- element is not as effective as the true wild-type and many strains have gradually darkening eye color over time (also, in some lines, one sex has darker eyes than another, but this is not consistent between strains, and in most strains you can t sex flies by eye color). The darker

5 6 SI eyed flies are generally older than the lighter eyed flies in such lines. Note: Look at the white eyed mutants while they are still in the vial please do not etherize them. 4. Look for any other apparent differences between your strain of flies and wild-type. Some things you should look for: what is the body color compared to wild-type, are the bristles on the dorsal (top surface) of the thorax as long as wild-type, is the shape of the wing different from wild-type. Note: These will not be the same for everyone. If you have questions, please ask the instructor. 5. Practice sexing your flies. Male flies have darkly pigmented claspers at the end of their abdomen. Female flies lack these claspers, and have a somewhat broader abdomen. Females are often larger than males. Try to separate the males and females in the flies you have been examining. Make two little piles of flies. When you think you have done this successfully, have the instructor come over to doublecheck your work. DO NOT PROCEED UNTIL YOUR SEXING SKILLS ARE APPROVED BY THE INSTRUCTOR. 6. Sketch the differences between male and female Drosophila. 6. Discard the flies you have been working with by dropping them into a morgue. A fly morgue is usually a bottle filled with 70% ethanol that dead or unneeded flies can be dropped into for a quick and painless disposal. 7. Etherize a few more flies from your transposon stock, and quickly separate 5-10 males. When you have done this, place the flies into a completely empty (no food) Drosophila vial, and then let your instructor know. Your instructor will provide you with a vial containing females from the mapping strain and will help you to combine the two vials together for the first cross. 8. Your instructor will tell you the names of the mutant loci in the mapping stock you will be using and will describe for you the phenotype of these mutations. Record the names and the phenotypes of each mutation as part of your journal. You will examine these phenotypes more carefully in two weeks. 9. If you have time at the end of lab, etherize and scan through any remaining flies in your vials and see if you can find any newly eclosed animals (newly emerged from the pupal case). These animals will be almost devoid of pigment. Can you sex these animals? Examine their posterior ends carefully. These newly eclosed animals will be virgins (fruit flies do not mate for several hours after the emerge from the pupa) this is important because Drosophila females can store sperm from a single mating over a long period of time, so if you want to be sure that a male of a particular genotype is the father of the progeny you are scoring, it is often desirable to start with virgin females. If you find any virgin females, let your instructor know you may be able to set up an additional cross using your virgin females and males from the mapping stock. (Note: the females from the mapping strain that were provided to you by the instructor were not virgin why does that not matter for this cross?)

6 7 SI 1. Journal of Results- Name: The stock number of my mutant line is The eye color of my mutant line is Other differences between my mutant line and wild-type (or white) Sketch differences between male and female Drosophila Names and phenotypes of the mutant loci in the mapping strain that we are using.

7 8 SI Lab 7. Bioinformatics: Biology in Silico Introduction Biological experiments have traditionally been described as being divided into two types. In vivo experiments are experiments that are conducted using a living organism. In vitro experiments are experiments that are conducted in the absence of a living organism many in vitro experiments can be described as test-tube experiments in which researchers try to reproduce the behavior of an in vivo experiment while using as few components as possible. Our chromatography experiment using fruit fly eye pigments might be considered as an in vivo experiment, while our analysis of the behavior of starch branching enzyme on an agar plate containing glucose-1-phosphate during the pea lab might be considered an in vitro experiment. Scientists have recently added a third alternative type of experiment in an in silico experiment, the entire experimental exercise is performed on a computer. This type of experiment has both advantages and disadvantages. The advantages include taking advantage of the powerful computational capabilities of modern computers, and their impressive ability to organize enormous amounts of data and search these large databases quickly and efficiently. One of the disadvantages of the in silico approach is that in silico experiments are generally limited to analyzing data that has already been collected by other methods. Extrapolating the results of in silico experiments to new situations for which traditional experimental have not been collected can be very risky. In addition, unfounded assumptions on the part of the researcher about how biological processes operate can be inadvertently built into in silico experiments such that the experiment can produce the expected results, not because a biological system actually works that way, but rather because the researchers have designed the computer algorithms to insure those results. This sort of circular logic can be very problematic, and any in silico research strategy is best accompanied by reality checks parallel in vivo or in vitro experiments. Bioinformatics is one kind of in silico approach to biology that has received an enormous amount of attention in the last few years. This is in large part because bioinformatics provides the computer architecture that allows for the assembly, analysis, and access to the many genome projects that have been completed over the last several years. Each genome project is based on the assembly of DNA sequence traces, which are rarely more than 1000 nucleotides long. These strings of 1000 nucleotides have to be matched up and assembled into much longer strings, which represent the DNA sequence along a chromosome. Once this is done, genes have to be identified in the strings, compared to genes known from other organisms, and close matches have to be catalogued. To perform each of these steps by eye would take an enormous amount of time and effort, and would be extremely error-prone. Using computer algorithms to perform each of these steps is not entirely error-free, but it greatly speeds up each of these steps. Between 1997 and 2003, over 150 genome projects were completed and the rate of genome sequencing continues to increase. Today in lab, we are going to be using bioinformatics tools to learn more about the Drosophila transposon mutations that we began to work with in the last lab. First, we will examine one fruit fly mutation together as a class, and then you will have time to begin to use

8 9 SI bioinformatics tools to study the mutation that you are genetically mapping in lab. The databases we will examine today are the results of an in silico analysis of Drosophila genes. The genetic crosses that we started in lab, and that we will continue over the next several weeks, are designed to serve as a reality check for the analyses conducted by these databases. Materials A computer with world wide web access. The stock number for your fruit fly mutation stock. Procedure 1. Open your web browser. The sites we will be visiting should work well with most common web browsers. 2. Type in: Note: This web site and all of the other web sites mentioned in the lab handout will be archived on the Web pages section of the course website on Blackboard. You can, if you wish, access these sites from the course page instead of typing in each web address. 3. The Flybase web site should appear on your screen. Click on the Stocks link. 4. At the search prompt, type in the number: This is a stock very much like the ones that you are studying in lab the only difference being that this one has already been genetically mapped. 5. When you hit return, the next screen will provide some information about stock 10391, including the names of all of the mutations that are found in this stock. Start to fill in the section of the worksheet at the back of this lab that relates to this stock. 6. On the back of page 3 is a diagram of the cytological bands found on the Drosophila chromosome on which both the transposon mutation carried in stock and the mutation carried by your stock are found. This type of chromosome band map is sometimes called a cytogenic or a cytogenetic map. By convention, divisions 1-20 are found on the X chromosome, divisions are found on chromosome 2, divisions are found on chromosome 3, and divisions are found on chromosome 4. Divisions are divided into subdivisions labeled with letters (A, B, C, etc), and subdivisions are further divided into bands labeled with numbers. So a typical band designation might be 63B4, for the 63 rd division, 2 nd subdivision, 4 th band. You should find and mark the location of the P element insertion in stock

9 10 SI 7. How do they know which band contains the P-element? The researchers who created this insertion used a technique called in situ hybridization to label the location of the insertion in the polytene chromosomes of the larval salivary gland. They then examined the labeled chromosome under the microscope, and the band containing the P-element appears darker than other bands. You will see some of these pictures in a few minutes. 8. Click on the insertion link near the bottom of the page. This will bring you to a page with many other links on it. You are interested in the links for associated genes. One of the associated genes is labeled Eco/lacZ, which is part of the transposon construct that was used, and is not of interest to us at the moment. Click on the other associated gene link. 9. The page that appears next provides you with some additional information about where this gene is located and what its role is in living fruit flies. Some of the information you have already found elsewhere, but here you see some new information including its map location on the recombination map and a diagram of the region of the chromosome in which the transposon mutation is found. For a close-up view, click on the cytogenetic map link the gene of interest should be circled in red. Identify some of the genes near the mutation of stock When you are finished, you can go back to the previous page, where you can find the link to the Drosophila genome scaffold a scaffold is a large section of a genome sequence that is used when the size of the total genome is too large to be conveniently contained in a single file. This link will bring you to a web site run by the National Center for Biotechnology Information or NCBI. To get to this site directly you can use the following link: It is by comparing these flanking sequences with the whole genome sequence that it is possible to know where exactly (down to the specific base pair) a transposon insertion is located in the genome. This section of scaffold has approximately 157,875 bases and also contains annotations concerning the location of many genes. 10. Go back to the previous page. The function of a gene is often determined in part by comparison with genes that have already been described. These are listed in a section called SIMILAR GENES. In the summary section of the screen, you can find out about some of the genes with which this gene interacts, and the tissues in which this gene is expressed. These are other important ways to learn about gene function. If you want additional information not found on this page, there are also links to lists of references that can provide additional information about the gene. 11. Now you re ready to start looking at the stock that you have been working with in previous labs. Go through the same set of steps that you went through to learn about stock and fill in the journal.

10 11 SI

11 12 SI 1. Journal of Results- Name: Stock Genotype (define what each of the symbols mean): P-Element Insertion site(cytogenetic Map) Location on the Recombination Map What is the name of the gene interrupted by the P-Element What is the gene similar to? What other genes are nearby? What does the gene product do? Now, take a look at the stock that you have been working with in lab. Stock Genotype (define what each of the symbols mean): P-Element Insertion site(cytogenetic Map) Location on the Recombination Map

12 13 SI What is the name of the gene interrupted by the P-Element What is the gene similar to? What other genes are nearby? What does the gene product do?

13 14 SI Lab 8. Transposon Mapping: Sorting the F1 Introduction It takes 10 days for wild-type Drosophila melanogaster to mature from egg to adult at 25 degrees C. It has now been over two weeks since we initiated our P0 cross, and it is now time to collect heterozygotes for the F1 backcross. We are going to collect only female heterozygotes because there is no recombination in male Drosophila, only in females. The reasons for this are obscure, but many organisms have different rates of recombination between males and females, and fruit flies are just an extreme case in which one sex has recombination while the other does not. We also have to collect virgin females this is because fruit fly females store sperm between matings, and will lay eggs that have been fertilized by different fathers. As you might expect, this would greatly complicate our analysis. By collecting virgin females, we will ensure that we the experimenters determine the paternity of the progeny that we will score in a couple of weeks. You can determine that a female Drosophila is virgin in a number of ways. One way is to look at the abdomen of a female Drosophila if the cuticle (the material that makes up the exoskeleton) is still soft and near white, and the larval gut tissue or meconium is still visible as a black spot slightly off the ventral midline of the abdomen, then the animal is virgin. Please note that newly emerged males are also pale white and have a meconium, so also be sure that the fly is female (remember, males have claspers at the end of their abdomen these are usually brown in color and darken before the rest of the cuticle). Another way of obtaining virgins is to use a timing method: that is flies will not mate for several hours after emerging from their pupal cases if you remove all of the flies from a vessel (called clearing ) and come back after a few hours, all of the animals in the vessel when you come back will have emerged recently, and can be considered virgin, even if they no longer are pale and no longer show a meconium. Your instructor will tell you which method we will be using. Not only are we only selecting females, and only virgin females, but we have to select virgin females of the correct genotype. Remember that because we are interesting in mapping the position of the transposon insertion, and because the transposon is marked with an eye color marker, the females we want will have pigmented eyes (yellow, brownish, orange, or red depending on the insert), and the ones we don t want will have white eyes. Remember also that males can also have orange eyes, but that males are not helpful to us. There may be additional markers segregating in this cross that may assist you in sorting progeny. Your instructor will help you determine what they are and how they can be helpful.

14 15 SI Overall Crossing Scheme Transposon line X white-eyed mutant mapping line Red eyed F1 progeny X white-eyed mutant mapping line (Test cross) Score backcross progeny Materials Drosophila, wild type and white mutants (for comparison with lines carrying P-elements) A unique Drosophila transposon insertion line for each student (acquired from the National Drosophila Stock Center at Indiana University in Bloomington, Indiana) A Drosophila stock that is mutant for the white locus and also carries several phenotypic markers that can be used to recombination map the transposon insert. The cross that you set up 2 weeks ago. Fly food Dissecting Microscopes Microscope Illuminator Etherizer and Re-etherizer Metal spatula and/or paintbrush Procedure 1. Your instructor will give you the same unique stock of Drosophila carrying a transposon insert that you examined two weeks ago. You and the person who shares your lab table with you will also have a vial of wild-type flies and a vial of the mapping strain for you to examine carefully under the microscope, in case you did not have the chance to do so in the previous lab. Etherize a few of each of these flies and look for phenotypic differences between them. If you feel uncertain of your ability to sex flies, you can sex these flies, and have the instructor come double-check you. Make sure you note as many of the mutant phenotypes of your unique stock as you can, and try to associate them with the genotype of your stock. 2. Last week, you learned about the genotype of your Drosophila stock using the on-line database Flybase. Using what you learned last week, diagram the cross that you set up 2 weeks ago, showing the genotypes of all of the progeny that you expect from this cross. 3. Identify the genotype and the phenotype of the individuals that you will use for the F1 test cross. These will be virgin female flies that you will mate to males of the mapping strain in order to produce the progeny that you will finally score in 2 weeks time.

15 16 SI 4. Now you are ready to examine the progeny of your cross from last week. Don t worry if there are only a few flies in your vial. Your instructor has been monitoring your crosses and may have already harvested some virgin females as insurance in case something happens to the flies you harvest today. Remember, you can only etherize and re-etherize these flies once because you don t want them to be sterile. Identify virgin females of the correct genotype, and put them in vial until your instructor has a chance to examine them and verify that they are virgin. If there are no flies in your vial at all, let your instructor know, and you will be provided with an alternative vial. The experiment has been designed so that all of the stocks will behave the same way at this stage, so even if it is a cross involving a different stock, it can be sorted the same way. Don t worry, you will be able to map your own stock in 2 weeks, even if you can t set up the cross today because of the flies your instructor has already harvested as insurance. Even if you aren t working with your own stock, you should still set up a cross, if possible, because you will be helping somebody else map his or her stock. 5. Your instructor will help you combine your females with males from the mapping stock to complete the cross. Make sure your name, the genotypes of the parents, and the date is written on the vial. Give this vial to your instructor.

16 17 SI 1. Journal of Results- Name: The stock number of my mutant line is The phenotypes exhibited by my transposon stock include: Diagram the genetic cross that we initiated weeks ago, showing the genotypes and phenotypes of both parents and of all classes of progeny from this cross: Identify the phenotypes and the genotypes of the progeny that we are collecting for use in the test cross (which is sometimes also called an F1 backcross):

17 18 SI Lab 10. Transposon Mapping: The Final Countdown! Introduction It is now time to score the testcross progeny. Unlike previous labs, in which we had to save some of the flies we were scoring, for today s lab, you can re-etherize your flies as often as you need until all of the flies have been scored. When you have scored all of your flies, they can be placed in the morgue. Remember, when you are scoring your flies that the male parent in the cross provides a chromosome containing only recessive alleles. Therefore, what determines whether you observe the white-plus transposon marker and whether you see the dominant or recessive phenotypes for the mapping loci, is the contribution of the female parent. For this reason, the scoring sheet included at the end of this packet only includes the female contribution to the offspring you are scoring because the male contribution is always the same and does not alter the observed phenotype. Overall Crossing Scheme Transposon line X white mutant mapping line Red eyed F1 progeny X white mutant mapping line (Test cross) Score backcross progeny Materials A Drosophila stock that is mutant for the white locus and also carries several phenotypic markers that can be used to recombination map the transposon insert. The cross that you set up 2 weeks ago Dissecting Microscopes Microscope Illuminator Etherizer and Re-etherizer Metal spatula and/or paintbrush Procedure 1. Take some wild-type and some mapping strain flies, etherize them, and examine them under the microscope. Make sure you can distinguish them from one another for each of the traits by which they are supposed to differ (wing veins, body color, etc.). Remember

18 19 SI that while the eye color between these two strains is very distinct, the eye color difference between your transposon strain and the white phenotype of the mapping strain may be much less so. 2. Once you have convinced yourself that you can distinguish each of the phenotypes, you can begin to score the progeny from your cross. You should keep in mind that some of the transposon strains carried a mutant yellow allele on the X chromosome, and as a result, some of the males among your progeny may have yellow body color. It is sometimes possible to see the effects of yellow and other body color phenotypes at the same time (e.g. yellow black double mutants are darker than yellow single mutants, but lighter than wild-type, you can ask your instructor for advice about how to distinguish between these alternatives), but if you are having trouble distinguishing between alternatives, you can just exclude all of the yellow males from your tallies. The reason why this is okay is that yellow segregates completely independently from the alleles on the mapping chromosome and disposing of these progeny should not bias your results. 3. It is recommended that you use a binary system for scoring the flies. First sort the flies by eye color. Then take the flies of one eye color and sort them by body color. Then take the flies of one eye color and body color, and sort them for wing phenotype. Then count the flies of that phenotype, using hatch marks grouped in fives to indicate how many flies you have counted in a given phenotype on your scoring sheet (see example sheet). You may find that making piles of 5 flies all of the same phenotype and counting piles of five is easier than counting flies one by one. Once you have done this for one phenotype, back up a step and count the other group, continuing to sort and count until you have scored all of the flies in the vial. 4. Many of you will have more than one vial of flies to count. If that is the case, use a separate reporting sheet for each vial. Your instructor will have more sheets for you to use. The reason for this is that in case of contamination (e.g. wild-type flies in one of your vials), you will only have to throw out the data from one vial rather than throwing out the data from all of the vials for that genotype. If you recorded data from all of the vials on the same data sheet, it would be difficult to tell which data came from which vial. 5. Count up the hatch marks for each phenotype for each vial, writing the number in the margin next to the appropriate phenotype. Calculate the total number of flies scored in each vial. 6. Finally, ask your instructor for another scoring sheet. Use this scoring sheet as a summary sheet, writing in the numbers of each phenotype you scored for each vial in the space where you had placed hatch marks before. Add the numbers for each phenotype up, and write the total for each phenotype in the margin. Add your totals for all of the phenotypes up to come up with a grand total for the number of flies you scored today. Double-check your math by adding up the totals for each of your individual vials to see if your number matches the grand total. If there is a discrepancy, do the math again. 7. Staple all of your data sheets together with the summary sheet on top and hand it in to the instructor. Be sure your name, the stock number, and the genotype of your strain is on each data sheet.

19 20 SI Name Stock # Date Genotype of transposon stock: Genotypes of test cross progeny (remember, all progeny will also be homozygous or hemizygous for the white mutation on the X chromosome: w/w or w/y; and will have a copy of the second chromosome with recessive curled and black mutations on it) c 1 b 1 c + b 1 c 1 b + c + b + c 1 P[lacW] b 1 c + P[lacW] b 1 c 1 P[lacW] b + c + P[lacW] b + Calculate the map distances between the P[lacW], curvy (c), and black (b), and draw a map showing the positions of each of the three genes.