The bioinformatic enhancement of exercises in Drosophila genetics.
The model system for the genetics laboratory exercise is the fruit fly, Drosophila melanogaster. The following required materials can be ordered from the Carolina Biological Supply Company (www.carolinabiologicalsupply.com):
* One vial of Drosophila stock culture of choice (either the F1 apterous x sepia, the F1 vestigial x sepia, or the F1 vestigial x ebony cross) for each set of ten students.
* One Drosophila BioKit for a class of up to 30 students.
* Either a 10X hand lens or a dissecting scope for each pair of students.
* Order the fruit fly stocks at least seven days prior to use to allow sufficient time for delivery. I typically order vials of the three different cultures to provide the class with some variety in this exercise.
* The Drosophila BioKit contains all the necessary materials (instant Drosophila medium, culture vials, vial plugs, FlyNap anesthetic kits, labels and sorting brushes, student guides, an instructors guide, and Carolina Drosophila manuals)
* The Carolina Drosophila Manual (Flagg, 1988) contains all the information regarding preparation of new Drosophila breeding vials with medium, manipulation of flies, and photographs of the phenotypes discussed in this paper.
* Either dissecting microscopes or 10X hand lenses can be used to view the fruit fly phenotypes. If students work as teams of two or three, I then ask one student to serve as an observer of phenotypes and the others to serve as data gatherers. Students can then rotate these assignments throughout the lab period.
Fruit Fly Lab Protocol
1. When the Drosophila stock cultures arrive, maintain them in a cool (room temperature) environment. Observe daily until approximately 20 or more adult files are visible; this typically occurs 3-5 days after receipt of the stock culture.
2. Each team of two students should set up three new vials of fresh Drosophila medium. Anesthetize the adult flies and, in each new vial, place three wild type males and three wild type females. Cap the new cultures and maintain at room temperature.
3. After 7-10 days, remove the six F1 adult flies from the new cultures and dispose of properly.
4. After approximately 2 weeks of incubation, new adult flies (the F2 generation) should now appear in the new cultures. Adult flies should be counted and assigned into separate phenotypic classes as designated in Table 3. These counts should begin during the first lab period after the appearance of adult flies, and should be continued for 10 days during assigned lab periods.
The fruit fly cross is a dihybrid cross involving two traits, each trait encoded by a single gene, each gene being represented by two alleles in that specific cross. There are three different crosses of this type available from Carolina Biological Supply Company. The first involves the mutant phenotypes vestigial (vg) wing and ebony (e) body, the second the traits apterous (ap) wing and sepia (se) eye color, and the third vestigial wing and sepia eye. I often utilize all three crosses in a single laboratory exercise to provide students with some variety in their efforts. The separate phenotypes and their respective genotypes are described in Table 1; the three separate dihybrid crosses are outlined in Table 2. The full laboratory protocol is outlined in the Fruit Fly Lab Protocol, and an example of a data table for student use is shown in Table 3.
* The appearance of adult flies in the stock cultures ordered from Carolina Biological Supply Company indicates that the cultures are ready for student manipulations in the next scheduled laboratory session. These emerging adult flies constitute the F1 generation and represent the breeding stock for the students' experiments.
* When collecting adult F1 flies to set up student crosses, there is no need to collect virgin flies, as these individuals for all three crosses are all of the same genotype (heterozygous).
* After F2 generation adult flies begin to appear in the vials in which the students have set up their crosses, collection of data should begin. Counts after 10 days might include flies from the next (F3) generation, and thus would contaminate the data set.
Questions for Students: Fruit Fly Lab
I set up each of the three experiments in the same manner. Students are not told a priori which experiment they are conducting, only that they are analyzing the results of one of the three possible Mendelian dihybrid crosses. Their goal is to determine which traits in their experiment are being controlled by specific genes. The students first determine which traits exhibit binary phenotypes (e.g., red eye vs. brown eye). They then catalog the two sets of phenotypes under consideration (e.g., normal vs. vestigial wing, red eye vs. sepia eye)
After the first round of data-gathering from the F2 generation that the students have set up, it quickly becomes evident to the students which traits are exhibiting binary (that is, two state) variation. To direct their inquiry, I ask them to determine which traits exhibit binary states in their particular crosses. The wing phenotypes are very obvious (normal vs. either vestigial or apterous). The body color is often the next trait to be differentiated by the students, the wild type yellow brown being rather distinct from the mutant ebony, in which the fly appears to be a very dark brown-to-black color. The eye colors are the most problematic, the wild type red being a very lustrous red color, and the sepia being a dull brown color. The best approach in determining the body and eye color phenotypes is to conduct a side-by-side comparison of several flies of contrasting phenotypes (e.g. red eye vs. sepia eye) under 10X magnification. Once students see the differences in phenotypes, they are more confident and reliable about making future phenotype designations.
The Fly Nap compound is harmless to humans, although a slight odor will be detected when the bottle is opened. It is best to keep the bottle tightly capped when not in use, as the odor is quite noticeable. After the fruit flies are counted, we dispose of them by first drowning the flies, then flushing them down the sink. This species of fruit fly is not a normal pest species in North America, but we prefer to follow proper biological containment procedures.
Links to National Standards
The genetics exercise outlined here supports the following National Science Education Standards (NRC, 1996):
* Heritable characteristics can be observed at molecular and whole-organism levels: in structure, chemistry, or behavior.
* The sorting and recombination of genes in sexual reproduction results in a great variety of possible gene combinations from the offspring of any two parents.
Questions for Students: Computer Lab
Upon completion of the Fruit Fly Lab, I ask the students the following questions:
* What are the functions of the genes/alleles whose phenotypes they have been studying? That is, what protein is encoded by their gene/allele of interest, and what is the cellular location and function of that protein?
* How many different alleles are currently known to exist for each gene under study? From this exposure it might appear that each gene contains only 2 alleles (wild type vs. mutant)--is that a valid statement? Or could each gene have a number of associated alleles?
* Are the gene products that are responsible for these fruit fly phenotypes unique to Drosophila, or are these genes also present in other organisms? That is, to what extent, if any, are the fruit fly genes under study in this exercise shared by other organisms?
To address these questions I direct the students to a bioinformatics approach using desktop computers in our teaching lab. Alternatively, as this effort takes place after the Drosophila Lab Investigation has been completed and all the data have been gathered, one could utilize a computer lab separate from the biology laboratory, perhaps in the school library or in a separate computer lab. All the online resources discussed below are fully accessible by the general public through any speed of Internet connection.
Bioinformatic Lab Protocol
1. Access the National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov.
2. Open the TaxBrowser (Taxonomy Browser) on the NCBI homepage.
3. Locate the link to Drosophila melanogaster. Open, then locate the link to Flybase: http:// flybase.net.
4. To access information regarding a specific mutant allele, activate the Search Genes hot link on the Flybase opening page. This will open up the FlyBase Gene Query Form.
5. In the query window of the FlyBase Gene Query Form, enter either the phenotype name in the Phenotype window or the allele symbol (not italicized) in the Symbol/synonym/ name window.
6. Submit query. A synopsis of the mutant allele (termed "Synopsis of Gene--) will then appear with a variety of links and further information regarding gene sequence, function, map position on the chromosome, etc.
7. To access a description of other organisms that share the Drosophila gene product of interest, go to Protein domains and activate the first hot link for the allele of interest. This link will then direct you to the Taxonomic Coverage page that shows the range of organisms with homologous proteins.
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To begin their search, I refer the students to the National Center for Biotechnology Information Web site (http://www.ncbi. nlm.nih.gov), and open the TaxBrowser (Taxonomy Browser) on the NCBI homepage. From the Taxonomy Homepage, students can locate the link to Drosophila melanogaster, and a set of External Information Resources. The most useful of these is FlyBase (http://flybase.net). On the homepage of this site is a search engine for individual genes that allows one to find the description of a gene's biochemical product, its cytological location, the number of known alleles, and a synopsis of the gene (Figure 1). This is all standard information that I require students to include in their written research report and oral presentation of their lab efforts.
To illustrate this approach consider the mutant wing allele vg, for vestigial wing. To obtain information on vg enter the initial Web page on Flybase, A Database of the Drosophila Genome (Figure 1).
The second data class on this page contains a search engine portal for genes. Activating the Search Genes hot link will direct the student to the FlyBase Gene Query Form (Figure 2).
Here it is most efficient to search using both the mutant phenotype and the allele symbol fields. In the query window, enter "vestigial wing shape" for Phenotype and "vg" (not italicized) for Symbol/synonym/name. Submitting this query will then direct the user to the FlyBase Report, in this case "A Synopsis of Gene vg" (Figure 3) is accessed.
The upper portion of the page contains some rather sophisticated genetic and biochemical information, including cytogenetic and transcript map data, recombination map position, information regarding encoded polypeptides, and information regarding gene ontology. Gene ontology is a standardized vocabulary that transcends species boundaries and describes how a gene product operates within the cellular environment. A summary paragraph within the synopsis integrates the genetic, biochemical, and developmental information into a form readily grasped by informed students.
Answers to the first two questions may be found in this summary. The summary contains information regarding gene function, such as mutant microtubule formation, the different phenotypes (54) and the known number of alleles for "vg" (396). In order to answer the third question, the student may find the recombination map for "vg" (2-67.0) listed under Genomic Organization at the top of the synopsis. The "2" represents the chromosome containing the gene and "67.0" is the map position. The synopsis may also include the number of genetic (epistatic) interactions (290) and a list of literature references (429). More sophisticated biological information such as the molecular function and cellular localization of the gene product, the gene product's amino acid sequences and structural domains, and the biological processes affected by the gene and its associated mutants are contained in this synopsis. In the "Reports" column one can also access a "Full Report," which is an expanded version of the summary report available on this synopsis page. This summary contains further detailed information and hot links that allow self-directed inquiry.
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The "GENE ONTOLOGY" component is the most useful for the student in terms of locating relevant biological information regarding the function(s) of the gene product(s). The specific biochemical properties (GO: Molecular function), the biological processes mediated by the encoded protein (GO: Biological process) and the cellular localization (GO: Cellular component) are all hot-linked to more detailed explanations. The final topic in "GENE ONTOLOGY" is perhaps among the more useful hot links. For vg, the first entry is "Tubulin," which identifies one gene product as the microtubule protein tubulin. Activating this hot link directs the user to the EMBL-EBI (European Molecular Biology Laboratory--European Bioinformatics Institute) Web page that contains information regarding the tubulin protein. A short summary paragraph on this page describes the biological function of tubulin and some of the phenotypes associated with this protein. These last two Web pages will bring the student into the realm of biochemistry and cell biology, clearly demonstrating the connections between these different disciplines in the life sciences. With these tools the students can begin to understand the cellular and biochemical basis for their Drosophila phenotypic mutants. For example, tubulin is a structural component of adult Drosophila wings, providing the wings with structural integrity. The mutant vg allele encodes an altered tubulin protein that fails to form properly during wing formation in the pupal stage, resulting in vestigial wings. Thus the experiment is brought full circle. The initial student contact with the experimental system was through visual observation of the mutant phenotypes, and the penultimate bioinformatics analysis is the determination of the cellular basis for the mutant phenotype.
Introduction of the Concept of Universal Descent
Finally, I introduce the core evolutionary concept of universal descent (of all extant life forms) from a common ancestor in an indirect way, by asking the students if the genes that they have studied in Drosophila also have homologs in three other organisms: the brewer's yeast (Saccharomyces cerevisiae), the roundworm (Caenorhabditis elegans), and humans. The "protein domain" hot link on the "Synopsis of Gene" homepage (Figure 3) allows access to the European Bioinformatics Institute Web site (http://www.ebi.ac.uk/interpro/) (Figure 4).
This site provides information regarding the protein domain selected, including a paragraph that describes the function of the protein, links to descriptions describing the cellular location, the process in which the protein is involved, and any cellular structures that the protein is associated with. There is also a link to the Protein Data Base (PDB) that provides structural models of the protein. At the bottom of this page is a utility called "Taxonomic Coverage," that leads students into an evolutionary inquiry.
For example, if a student chooses the protein domain "Tubulin" from the "Synopsis of Gene vg" homepage, the Taxonomic Coverage diagram shows the number of tubulin protein matches in represented organisms such as Saccharomyces cerevisiae (4), Caenorhabditis elegans (19) and human (50) (Figure 3). Nineteen additional taxonomic groups are also shown, all (except viruses and an unclassified group) that contain protein domains with homology to the tubulin domain. This diagram is a simple representation showing the evolutionary links between the model organisms, demonstrating the fact that the protein domain of a Drosophila protein is also represented across a wide phylogenetic range of eukaryotes. I then ask students how this could be so, and after considering a number of conjectures, we typically arrive at a phylogenetic explanation. I introduce them to Erasmus Darwin's prescient conjecture in his book Zoonomia, in which he postulates the origin of all extant life forms from a single ancestor in deep time (Darwin, 1794), and ask them to consider whether their bioinformatics analyses on the five different Drosophila genes supports Darwin's suggestion. In this effort we engage in a class discussion, as different students have conducted analyses on different genes. To conclude the inquiry into the shared genetic homology, we examine the results of a comparative genomic analysis among Drosophila, Saccharomyces, and Caenorhabditis (Rubin et al., 2000). In this study, global comparisons were made among the protein domains of the known proteins encoded by these genomes, demonstrating shared homology for several thousand protein domains.
Links to National Standards
This bioinformatic exercise, by bringing into the picture an evolutionary perspective and demonstrating the relatedness of the fruit fly with other organisms, in terms of sharing common genes and proteins, connects with the following National Science Standards (AAAS, 1993; NRC, 1996):
* Molecular evidence substantiates the anatomical evidence for evolution and provides additional detail about the sequence in which various lines of descent branched off from one another.
* Life on earth is thought to have begun as simple, one-celled organisms about 4 billion years ago. During the first 2 billion years, only single-cell microorganisms existed, but once cells with nuclei developed about a billion years ago, increasingly complex multicellular organisms evolved.
* The millions of different species of plants, animals, and microorganisms that live on earth today are related by descent from common ancestors.
The effectiveness of this exercise is multiple. First, the hands-on laboratory activity is directly linked to a bioinformatic activity that is easily accessible from any networked computer. The students see the computer as an accessory to the laboratory investigation. Second, the student is able to answer questions through a guided inquiry into various databases. This builds student confidence, in that they are able to answer their own questions with appropriate guidance. Third, the biological basis for the Drosophila mutant phenotypes moves from the abstract to the tangible, in that a connection between a mutant protein and a physical trait is established. Fourth, the connections between cell biology, genetics, and biochemistry become selfevident. And finally, the students see for themselves the power of evolutionary biology in explaining patterns of global protein domain similarity among different organisms.
Appreciation is extended to Ms. Jackie Hucul for her review of the manuscript. Support from the NIH/INBRE (Award # 1P20 RR16481-02) is gratefully acknowledged.
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PATRICK J. CALIE (firstname.lastname@example.org) is Professor; SHARON LEE and EMILY JEAN HICKS are undergraduate students, all in the Department of Biological Sciences, Eastern Kentucky University, Richmond, KY 40475.
Table 1. Description of phenotypes seen in the Drosophila experiments. The traits shown below are each encoded by two alleles of a single gene, specific for that particular trait. The wild type phenotypes are encoded by dominant alleles, the mutant phenotypes by recessive alleles. Images to accompany each phenotype can be found in the Carolina Drosophila Manual. BODY COLOR WING SHAPE EYE COLOR Wild type = normal ([ap.sup.+] or [vg.sup.+]) Wild type = yellow Mutant = apterous Wild type = red brown ([e.sup.+]) (ap) ([se.sup.+]) Mutant = ebony (e) Mutant = vestigial Mutant = sepia (se) (vg) Table 2. Outline of the three Drosophila crosses presented in this exercise. Allele symbols are defined in Table 2. Parental phenotypes/ F1 generation F2 generation genotypes genotypes/phenotypes genotypes/phenotypes (not seen by students) (flies first seen by (offspring from students) student crosses) Apterous wing, red eye Red eye, normal wing 9 red eye, normal wing ap ap / [se.sup.+] [se.sup.+] se / s[e.sup.+] --/ ap+ -- [se.sup.+] [ap.sup.+] ap red eye, apterous wing X s[e.sup.+] --/ ap ap Normal wing, sepia eye sepia eye, normal wing [ap.sup.+] [ap.sup.+] se se / [ap.sup.+] -- / se se 1 sepia eye, apterous wing se se / ap ap Vestigial wing, red Red eye, normal wing 9 red eye, normal wing eye vg vg / [se.sup.+] se / [se.sup.+] -- / [se.sup.+] [se.sup.+] [vg.sup.+] vg [vg.sup.+] -- X 3 red eye, vestigial Normal wing, sepia eye wing [vg.sup.+] [vg.sup.+] [se.sup.+] -- / vg vg / se se 3 sepia eye, normal wing se se / [vg.sup.+] -- 1 sepia eye, vestigial wing se se / vg vg Vestigial wing, yellow Yellow brown body, 9 yellow brown body, brown body normal wing normal wing vg vg / [e.sup.+] [e.sup.+] e / [e.sup.+] -- / [e.sup.+] [vg.sup.+] vg [vg.sup.+] -- X 3 yellow brown body, Normal wing, ebony vestigial wing body [e.sup.+] -- / vg vg [vg.sup.+] [vg.sup.+] 3 ebony body, normal / e e wing E e / [vg.sup.+] -- 1 ebony body, vestigial wing e e / vg vg Table 3. An example of a data table for student use. Data for the second and third columns will be obtained by student observation in this lab; information regarding parental genotypes in the first column will need to be obtained by logical deduction, i.e., what must the original parental flies phenotypes (first column) have been in order to have obtained the observed results? Phenotypes/genotypes Phenotypes/genotypes Numbers of offspring of original of flies used to set in each phenotypic/ parental flies? up our genetic genotypic class from cross? our genetic cross?
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|Title Annotation:||INQUIRY & INVESTIGATION|
|Author:||Calie, Patrick J.; Lee, Sharon; Hicks, Emily Jean|
|Publication:||The American Biology Teacher|
|Date:||Oct 1, 2007|
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