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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 Blanco eye Forked bristle Shaven body White eye yellow chromosome Many genes Genes on DNA w1 w2 w3 Mutations in white b1 b2 1 Mutations in blanco 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. - Ornithine Citruline Enzyme1 Arginine Enzyme2 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 2 things out! Side bar: Naming mutants The wildtype eye color in flies is red Say two mutants are ISOLATED BY TWO DIFFERENT LABS Mutant flies have white eyes. The researcher who identified the first white eyed mutant lived in the US and named it white. Small case w designates the recessive mutant allele Upper case W designates normal (dominant) wildtype The researcher who identified the second mutant lived in Spain and named it blanco. Small case b designates recessive mutant allele Upper case B designates normal (dominant) wildtype allele The researcher who isolates the mutant names it!!!!! Cheapdate Cockeye king tubby Sevenless Bride of sevenless Daughter of sevenless 3 This process might also identify multiple mutants for the steps in the pathway! B----> E----> A----> N Mut3 Mut1 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 Mut3 Mut1 mut4 Mut2 4 White and Blanco QUESTION Are the two independently isolated mutations THAT HAVE THE SAME WHITE EYE PHENOTYPE disrupting the same or different genes? Precursor (white) Product (red pigment) Enzyme1 Gene1 Blanco=White???? OR Precursor (white) Intermediate (white) Enzyme1 Gene1 White Product (red pigment) Enzyme2 Gene2 Blanco 5 White gene singed forked Shaven body blanco white yellow 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! singed Blanco forked Shaven body white yellow 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. Mapping genes takes lots of crosses and is time consuming There is an easier way! 6 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: 7 True breeding Blanco x true breeding white The actual Cross Cross white (w) x blanco (b) w/w x b/b White Female Blanco male 8 If White and Blanco are the same gene …. Precursor (white) Product (red pigment) Enzyme1 forked blanco white yellow White= Blanco wwww (white eye) x wbwb (white blanco eye) F1 wwwb Phenotype= ????? In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All white ww wb 9 wwww (white eye) x wbwb (white blanco eye) F1 wwwb Phenotype= ????? In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All white ww wb 10 If White and Blanco are two genes…… Precursor (white) Intermediate (blanco) Enzyme1 Enzyme2 blanco Gene2 Blanco white forked Gene1 White yellow Product (red pigment) wwwwBB (white eye) x WWbb F1 Www Bb phenotype= ????? (blanco eye) 11 In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All red wwwwBB (white eye) x WWbb F1 Www Bb phenotype= ????? (blanco eye) In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All red ww B W bb 12 The cross WHITE EYE What are genotypes and phenotypes of the cross ww x bb b w w b (?) What is the eye-color of the w/b fly? ?White or Red???? 13 Precursor White -------> enzymeA product red White x white aa aa F1 aa White What happens if you do a self cross with the F1 a a aa (white) 14 Gene interactions give 9:7 Precursor----> intermediate----> white white EnzA product red EnzB White aaBB x F1 AaBb (phenotype= red) AB white AAbb Ab aB ab AB AABB AABb AaBB AaBb Ab AAbB AAbb AabB Aabb aB ab aABB aAbB aABb aAbb aaBB aabB aaBb aabb 9 A-B3A-bb 3aaB1aabb red white white 15 white 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. What if the w and b mutations disrupt the same gene? w b w b w F1 white=blanco b If the F1 w/b flies are white-eyed, we know that the white and blanco mutations disrupt one gene. What if w and b mutations disrupt two genes? w B+ W+ b w B+ W+ b F1 W+ b w B+ If the F1 w/b flies are red-eyed, we know that the white 16 and blanco mutations disrupt two genes. Single gene 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. singed forked Shaven body blanco white yellow If there is a single gene then ****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 w1 Blanco becomes w2 or wb 17 Precursor----> White product Red EnzA P aa F1 aa F2 x aa a a aa 18 Two genes 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 Say in the complementation test you get red eyed females 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) 19 Two genes 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 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 Product red Intermediate white Enzyme1 Enzyme2 gene1 W+ b forked blanco white singed B+ yellow blanco w Shaven body white gene2 20 Gene interactions give 9:7 Precursor----> intermediate----> white white EnzA product red EnzB White aaBB x F1 AaBb (phenotype= red) AB white AAbb Ab aB ab AB AABB AABb AaBB AaBb Ab AAbB AAbb AabB Aabb aB ab aABB aAbB aABb aAbb aaBB aabB aaBb aabb 9 A-B3A-bb 3aaB1aabb red white white 21 white Precursor white Product red Intermediate white Enzyme1 Enzyme2 gene1 gene2 OR Precursor white Product red Intermediate white Enzyme1 gene2 Enzyme2 gene1 22 Molecular basis of mutations 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? 23 Molecular basis of mutations 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 CCCCCCCCCCCCCCCCCCC = GeneW = proteinW The nucleotides in the sequence of the gene is critical for its proper function. 24 Mutant genes Nucleotide sequence of the normal W gene: ---CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-----GGGGGGGGGGGGGGGGGGGGGGGGGGGGGG--The sequence of the w1 mutation of gene W ---CCCCCCCCCCCCCCCCCCCCCCCCCCTCCC-----GGGGGGGGGGGGGGGGGGGGGGGGGGAGGG--- The sequence of the w2 mutation of gene W ---CCCCGCCCCCCCCCCCCCCCCCCCCCCCCC-----GGGGCGGGGGGGGGGGGGGGGGGGGGGGGG--- So at the molecular level, the w1/w2 white female fly would be depicted as: w1: ---CCCCCCCCCCCCCCCCCCCCCCCCCCTCCC-----GGGGGGGGGGGGGGGGGGGGGGGGGGAGGG--w2: ---CCCCGCCCCCCCCCCCCCCCCCCCCCCCCC-----GGGGCGGGGGGGGGGGGGGGGGGGGGGGGG--25 Two White Genes Lets make things more complicated. w1 and w2 disrupt one gene (geneA). w3 disrupt a second gene (geneB) Precursor white Product red Intermediate white Enzyme1 Enzyme2 Gene A Gene B Aw1 Aw2 Bw3 Disruptions in geneA (Aw1 and Aw2) and geneB (Bw3) give rise to white eyes. HOW DO YOU FIGURE OUT THAT w1 and w2 disrupt gene A and w3 disrupts geneB 26 Intermediate white Blanco enzyme Product red blanco White enzyme white Precursor white w1/w1 B/B w2/w2 B/B F1= w1/w2 B/B 27 Intermediate white Blanco enzyme Product red blanco White enzyme white Precursor white w1/w1 B/B W/W b(w3)/b(w3) F1= W/w1 b(w3)/B 28 Genes Precursor white Product red Intermediate white Enzyme1 Enzyme2 Gene A Gene B Disruptions in geneA and geneB both give rise to white eyes. Cross a Aw1 fly with a Aw2 fly and see if you get red eyes. If w1 and w2 disrupt geneA, they will/will not complement . What about Bw3? 29 Complementation analysis Genotype eye color Aw1/Aw2 Aw1/Bw3 Aw2/Bw3 white red red complementation N Y Y w1, w2 = geneA= complementation groupA (multiple alleles) w3,= geneB= complementation groupB 30 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 31 Complementation crosses We systematically perform crosses First we perform the cross d1/d1 x d2/d2 F1 d1/d2 are produced wing= flat or delta If they are flat, they disrupt -------- gene Then we perform d1/d1 x d3/d3 F1 d1/d3 wing=flat or delta You construct a complementation table + flat wing Mutation complement Different genes - is delta wing mutation don’t complement same gene 32 Complementation crosses You construct a complementation table + is flat wing Mutation complement Different genes d1 d2 d3 d4 d5 d1 + + - - is delta wing mutation don’t complement same gene d2 + + - d3 + + + d4 + + + d5 + + - Gene1= alleles (d1, d2, d5) Gene2= alleles (d3, d4) 33 The pathway Precursor delta Product flat Intermediate delta Enzyme1 Enzyme2 Gene1 Gene2 (allele d1, d2, d5) (allele d3, d4) 34 35
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