1 Lecture 10: What is a gene? Each chromosome has one DNA molecule Each chromosome has many genes A gene produces a protein that give rise to a phenotype A gene has many forms- alleles Different alleles are caused by different changes in the same gene Mutations in different genes CAN give you the same phenotype chromosome yellow Blanco eye Shaven body Forked bristle White eye Many genes Genes on DNA w1w2w3 Mutations in white b1b2 Mutations in blanco

2 Complementation The complementation test is a rapid method of determining whether two independently isolated mutants with the same phenotype (in the same pathway) are in one or two (or more) genes. OrnithineCitrulineArginine Enzyme1Enzyme2 Both mutant1 and mutant2 cannot make arginine. If you did not know the pathway you would wonder if these two mutants were mutations in the same gene or mutations in two different genes If you are working with Neurospora, you can feed the intermediate (Citruline) to the mutants and see if they can now make arginine. You are “complementing” the mutants with intermediates Mutant1+ citruline=cell makes arginine Mutant2+citruline=cell cant make arginine- mutant It is not often this easy. The wildtype eye color in flies is red Say two different laboratories isolated mutants in that had white eyes. You cant feed flies eye color precursor to figure things out! Glutamic acid- 

3 This process might also identify multiple mutants for the steps in the pathway! B---->E---->A---->N Mut3Mut1 mut4 Mut2 mut1 and mut4 might be two different mutations in the same gene. OR Mutations in two different genes that work in the same pathway B---->E---->S---  A---->N Mut3Mut1Mut2mut4

4 White and Blanco-one or two genes? Precursor (white) Intermediate (white) Product (red pigment) Enzyme1Enzyme2 Gene1 White Gene2 Blanco Precursor (white) Product (red pigment) Enzyme1 Gene1 Blanco=White???? OR QUESTION Are the two independently isolated mutations THAT HAVE THE SAME WHITE EYE PHENOTYPE disrupting the same or different genes?

5 Easy way!!! There is an easier way QUESTION: IS BLANCO THE SAME GENE AS WHITE or IS BLANCO AND WHITE TWO DIFFERENT GENES The following cross is performed: True breeding Blanco x true breeding white

6 The actual Cross Cross white (w) x blanco (b) WhiteBlanco Femalemale w/wxb/b

7 If White and Blanco are the same gene …. Precursor (white) Product (red pigment) Enzyme1 White= Blanco wwwwxwbwbwwwwxwbwb (white eye)(white blanco eye) F1w w w b Phenotype= ????? In the F1 will the flies be red eyed or white eyed? All white

8 The cross b w w b (?) What is the eye-color of the w/b fly? ?White or Red???? WHITE EYE What are genotypes and phenotypes of the cross wwxbb w wbwb

9 Precursor >product WhiteenzymeAred White x white aa F1 aa White What happens if you do a self cross with the F1 a a aa (white) F2

10 If White and Blanco are two genes…… Precursor (white) Intermediate (blanco) Product (red pigment) Enzyme1Enzyme2 Gene1 White Gene2 Blanco w w w w BBxWWbb (white eye)(blanco eye) F1Ww w Bb phenotype= ????? In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All red

11 w w w w BBxWWbb (white eye)(blanco eye) F1Ww w Bb phenotype= RED In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All red w b W B

12 Gene interactions give 9:7 Precursor---->intermediate---->product whitewhitered EnzA EnzB AB Ab aB ab AB Ab aB ab AABBAABb AaBB AaBb AAbB AabB aABB aAbB aABb 9 A-B-red 3A-bbwhite 3aaB-white 1aabbwhite AAbb Aabb aaBB aaBb aAbb aabB aabb Whitex white aaBBAAbb F1AaBb (phenotype= red) What happens if you do a self cross with the F1 F2

13 Map Genes Mapping genes takes lots of crosses and is time consuming There is an easier way! yellow singed Shaven body forked white Blanco If on the other hand the two mutations map to different regions of the chromosome (or different chromosomes) then that would indicate that they are two different genes. You could map each mutation. If Blanco = white then the two mutations WILL map to the same spot on the chromosome. That would indicate that they are the SAME GENE -two different alleles! yellow singed Shaven body forked whiteblanco

14 How many genes? The answer to the question (What is the eye-color of the w/b fly?) depends on whether the w and b mutations disrupt the same gene or two different genes. If the F1 w/b flies are red-eyed, we know that the white and blanco mutations disrupt two genes. What if the w and b mutations disrupt the same gene? white=blanco w b w w b b W+b wB+ F1 wB+ w W+b b What if w and b mutations disrupt two genes? If the F1 w/b flies are white-eyed, we know that the white and blanco mutations disrupt one gene. F1

15 If the F1 flies are white-eyed, the mutations disrupt the same gene. A geneticist would say the two mutations do not complement one another because normal function is not restored. Single gene If there is a single gene then yellow singed Shaven body forked whiteblanco ****Nomenclature**** If the researchers discover that blanco (b) and white (w) are mutations within the same gene, there is a problem. The same gene has two names. One would like to have names that indicate that these are two alleles of the same gene. These are renamed White becomes w 1 Blanco becomes w 2 or w b

16 Two genes Say in the complementation test you get red eyed females There are two genes --- W and B. I f there are two genes then: How do these two genes relate to one another? (one gene one enzyme) If on the other hand the w/b F1 flies are red-eyed, we know that the white and blanco mutations disrupt two genes. Geneticist would say that these two mutations complement one another. They complement because normal function is restored

17 Two genes There are two genes --- W and B. If there are two genes then: How do these two genes relate to one another (one gene one enzyme) Precursor white Intermediate white Product red Enzyme1Enzyme2 gene1gene2 white blanco wB+ W+b If the F1 w/b females are red-eyed, then white and blanco mutations disrupt two genes. You say that these two mutations complement one another. They complement because normal function is restored yellow singed Shaven body forked white blanco

18 Enzyme1Enzyme2 Precursor white Intermediate white Product red gene1gene2 Enzyme1Enzyme2 Precursor white Intermediate white Product red gene2gene1 OR

19 Now what is the molecular basis for two mutations within the same gene? Lets say that w1 and w2 both disrupt geneW What is a gene? It’s a piece of DNA with a specific sequence DNA consists of a linear array of the four nucleotides Adenine- Cytosine- Guanine- Thymine ACGT Specific DNA sequence = gene = protein ATGCCCCCCCCCCCCCCCCCCCTAA = GeneW = proteinW The nucleotides in the sequence of the gene is critical for its proper function. Molecular basis of mutations

20 Mutant genes Nucleotide sequence of the normal W gene: ---ATGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTAA TACGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGATT--- The sequence of the w1 mutation of gene W ---ATGCCCCCCCCCCCCCCCCCCCCCCCCCCTCCCTAA TACGGGGGGGGGGGGGGGGGGGGGGGGGGAGGGATT--- The sequence of the w2 mutation of gene W ---ATGCCCCACCCCCCCCCCCCCCCCCCCCCCCCCTAA TACGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGATT--- So at the molecular level, the w1/w2 white female fly would be depicted as: ---ATGCCCCCCCCCCCCCCCCCCCCCCCCCCTCCCTAA TACGGGGGGGGGGGGGGGGGGGGGGGGGGAGGGATT ATGCCCCACCCCCCCCCCCCCCCCCCCCCCCCCTAA TACGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGATT---

21 Two White Genes Precursor white Intermediate white Product red Enzyme1Enzyme2 Gene A A w1 A w2 Gene B B w3 Disruptions in geneA (A w1 and A w2 ) and geneB (B w3 ) give rise to white eyes. HOW DO YOU FIGURE OUT THAT w1 and w2 disrupt gene A and w3 disrupts geneB Lets make things more complicated. w1 and w2 disrupt one gene (geneA). w3 disrupt a second gene (geneB)

22 Genes Precursor white Intermediate white Product red Enzyme1Enzyme2 Gene A Gene B Disruptions in geneA and geneB both give rise to white eyes. Cross a A w1 fly with a A w2 fly and see if you get red eyes. If w1 and w2 disrupt geneA, they will/will not complement. What about B w3 ?

23 white blanco White enzyme Blanco enzyme Precursor white Intermediate white Product red w1/w1 B/B w2/w2 B/B F1= w1/w2 B/B

24 white blanco White enzyme Blanco enzyme Precursor white Intermediate white Product red w1/w1 B/B W/W b(w3)/b(w3) F1= W/w1 b(w3)/B

25 Complementation analysis Genotypeeye color complementation Aw1/Aw2whiteN Aw1/Bw3redY Aw2/Bw3redY w1, w2 = geneA= complementation groupA (multiple alleles) w3,= geneB= complementation groupB

26 Suppose we isolate 5 delta wing mutations d1 d2 d3 d4 d5 We want to know how many genes are disrupted in these mutations and which mutations are in the same complementation group

27 Complementation crosses We systematically perform crosses First we perform the cross d1/d1xd2/d2 F1d1/d2 are produced wing= flat or delta If they are flat, they disrupt gene Then we perform d1/d1xd3/d3 F1d1/d3 wing=flat or delta You construct a complementation table + flat wing- is delta wing Mutation complementmutation don’t complement Different genessame gene

28 Complementation crosses You construct a complementation table + is flat wing- is delta wing Mutation complementmutation don’t complement Different genessame gene Gene1= alleles (d1, d2, d5) Gene2= alleles (d3, d4) d1d2d3d4d5 d d d d d5--++-

29 The pathway Enzyme1Enzyme2 Precursor delta Intermediate delta Product flat Gene1 (allele d1, d2, d5) Gene2 (allele d3, d4)