Biology 234 J. G. Doheny Chapter 12 (Part III) Complementation Analysis You can use a Genetic Dissection analysis to find which genes and which proteins are involved in a biochemical process. (For example, how many proteins are involved in forming the Drosophila Barrier-Insulator complex, that prevents heterochromatin from spreading into euchromatin.) A complementation analysis is something you have to do when you are doing genetic dissection. If you do a random mutagenesis screen you will create several different mutants, but you might have mutated the same gene more than once. A complementation analysis allows you to tell the difference between two mutations to the same gene and two mutations to two different genes. This way you can tell how many genes (and how many proteins) are involved in the biochemical process. (For example, how many genes form the Drosophila Barrier-Insulator.) Why you do a Genetic Dissection Analysis: To find out what genes (and therefore what proteins) are involved in a biochemical process. (For example: how many proteins form the Barrier-Insulator Complex in Drosophila?) Why you do a Complementation Analysis: To find out how many genes (and therefore how many proteins) are involved in the biochemical process. This is necessary because you start a Genetic Dissection Analysis by doing random mutagenesis screen in order to generate mutant strains of Drosophila. But you will probably mutate the same genes several times. Therefore, how do you tell the difference between two different mutations to the same gene, and two different mutations to two different genes? A Complementation analysis allows you to do this. A Random Mutagenesis Screen generates Dominant Negative Mutations: Mutations can be either dominant or recessive. The Wild-Type version of a gene is usually defined as the functional version of the gene, while a mutant version is the non-functional version. A functional version of a gene is usually dominant to a non-functional version (for example, the brown eye gene is dominant to the blue eye gene). However, there is a type of mutation called an antimorphic mutation, where the mutant version of the gene is dominant to the Wild-Type gene. That means that, if you have just one copy of the mutant version of the gene you will get the mutant phenotype, and having a Wild-Type version of the gene will not help. Antimorphic mutations are said to be Dominant Negative, because the mutant version of the gene is dominant over the Wild-Type version of the gene. When you do a random mutagenesis screen, and look for mutant phenotypes, the screen will favor finding Dominant Negative mutations, because the odds of mutating BOTH copies of the gene are extremely low. (The random mutagenesis screen may have generated many recessive mutations, but you d never see them because you would have to mutate BOTH copies of the gene in order to see the mutant phenotype.) Thus, when you do a random mutagenesis screen, and look for mutant phenotypes, the screen will preferentially find Dominant Negative mutations. 1
Dominant Negative Mutations are usually Homozygous Lethal: Antimorphic (dominant negative) mutations are generally very disruptive types of mutations. They have to be in order to give you a mutant phenotype, even when there is only one mutant copy of the gene present. They are usually so disruptive, in fact, that if you have TWO antimorphic mutations present, the organism will not survive long enough to be born. (They are said to be homozygous lethal.) For example, the mutant Fibroblast Growth Factor Receptor that gives rise to Achondroplasia (dwarfism) is a dominant negative mutation which is homozygous lethal. If you have two Wild- Type copies of the gene you will be normal height. If you have one mutant copy of the gene you are a dwarf. If you have two copies of the mutant version of the gene you will not survive to be born, and will be stillborn. The mutant allele of the Huntintin protein gene that gives rise to Huntington s Disease is also a dominant negative mutation that is homozygous lethal. Thus, people who develop Huntington s Disease are heterozygotes. Thus, organisms that carry two copies of an antimorphic, dominant negative mutation usually die before birth. Dominant Negative mutations are usually Homozygous Lethal. Dominant Negative Mutations are (usually) Denoted As A Lower-Case Letter With A d Superscript: The Wild-Type allele of a gene is denoted with an italicized capital letter (for example A) A dominant negative allele would be denoted as a d. The Theory Behind Complementation Analysis: If you do a random mutagenesis screen (in Drosophila, for example) you will generate mutant strains of flies that are carrying ONE dominant negative mutant copy of a gene. If you cross together two heterozygous strains of flies that are both carrying a mutation of the same gene, some of the offspring will get TWO copies of the same mutant allele and die. On the other hand, if you cross together two heterozygous strains of flies that are carrying mutations to two DIFFERENT genes, all of the offspring will be viable (will survive). So the two mutations are said to complement one another. For example, the following cross will complement: PARENT FLIES: Aa d ; BB X AA; Bb d (The parents carry heterozygous mutations to two DIFFERENT genes. One strain is carrying a dominant negative a d mutation and the other is carrying a dominant negative b d mutation) Offspring: AA; BB (viable) Aa d ; BB (viable) AA; Bb d (viable) and Aa d ; Bb d (vable) However, the the following cross will fail to complement, and one quarter of the offspring will be non-viable. (There will be a reduction in the number of offspring.) 2
PARENT FLIES: A ad ; BB X A ad ; BB Offspring: AA; BB (viable) Aa d ; BB (viable) a d a d ; BB (NOT VIABLE!) How You Carry Out A Complementation Analysis: Do a random mutagenesis screen to find mutant phenotypes. Then cross all of the strains to each other (one at a time), and make note of any crosses that give you significantly fewer than the expected offspring. Assume that any crosses that give you a reduced number of offspring are actually mutations to the same gene. Examples of Complementation Analysis Problems: Example 1: You want to find out how many proteins form the Barrier-Insulator complex in Drosophila melanogaster. You use the wm4 inversion as a reporter gene, do a random mutagenesis screen by feeding female flies EMS (a mutagen), and then generating three different strains of E(var) mutations. From the complementation analysis data below, how many genes, and how many proteins form the Drosophila Barrier-Insulator complex? (Answer: two (1,3) and (2)) 3
Example 2: You want to find out how many genes (proteins) are involved in eye formation in the model organism Zebrafish (Danio rerio). You do a random mutagenesis of fish, and generate 5 strains of fish that do not have eyes (five eyeless mutant strains). You cross them together and get the following results. How many genes are involved in Zebrafish eye development? (Answer: three genes (1,3,5) (2) and (4)) 4
Example 3: Vitis vinifera is a species of grapes used to make red wine. These grapes contain three types of sugar: glucose, sucrose, and fructose. You wish to know which genes (proteins) are involved in making fructose (how many genes are involved in fructose anabolism). You do a random mutagenesis screen, and generate 9 strains of grapes that do not make fructose. You do a complementation analysis. How many genes (proteins) are actually involved in making fructose in these grapes? (Answer: four genes (1,2,3) (4,5,6) (7,8) and (9) ) Practice Questions: Answer the following questions with one or two sentences. 1. What do you call a mutant allele of a gene that causes a mutant phenotype to be seen even though a Wild-Type allele of the gene is also present? (Two possible answers.) 2. Do random mutagenesis screens tend to generate dominant or recessive mutant alleles of genes? 3. Do random mutagenesis screens tend to favor the generation of dominant or recessive genetic mutations? Which. 4. Name two human genetic diseases that are caused by dominant negative mutations. 5. Name two human genetic diseases where the mutant allele is homozygous lethal. Be able to answer the following questions in one or two paragraphs: 1. What is an antimorphic mutation? 2. What is a dominant negative mutation? 3. What is a complementation test? 4. What is a complementation analysis used for? 5
5. Explain why random mutagenesis screens tend to generate dominant negative mutations? 6. What does the term homozygous lethal mean? 7. What percentage of the offspring will be non-viable: Aa d ;BB X AA; Bb d 8. What percentage of the offspring will be non-viable: Aa d ;BB X Aa d ; BB 9. What is a reciprocal cross? Be able to explain the following in one hand-written page or less: 1. Random mutagenesis screens create both dominant and recessive mutations, but only the dominant mutations are seen. Explain why. 2. What is a Complementation Analysis, what is it used for, and how do you do it? (You can use a specific example, like finding out how many proteins comprise the Drosophila Barrier-Insulator complex.) Suggested Problems from the Text Book: Chapter 6, Problem 77. 6