1. 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 mutations in the same gene Mutations in different genes can give you the same phenotype yellow blanco Shaven body Forked bristle white w1w2w3 chromosome Many genes Genes on DNA Mutations in a gene

2. 2 Complementation The complementation test is a rapid method of determining whether two independently isolated mutants with the same phenotype are in the same or different genes. OrnithineCitrulineArginine Enzyme1Enzyme2 Both mutant1 and mutant2 cannot make arginine. Are these two different 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. Mutant1+ citruline=arginine Mutant2+citruline=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!

3. 3 Naming mutants It is not often this easy. The wildtype eye color in flies is red 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

4. 4 White and Blanco 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 disrupting the same or different genes.

5. 5 White gene Mapping genes takes lots of crosses and is time consuming There is an easier way! If the two mutations map to different regions of the X- chromosome then that would indicate that they are two different genes. You could map each mutation. If the two mutations map to The same regions of the X-chromosome then that would indicate that they are the SAME GENE

6. 6 Easy way!!! There is an easier way The following cross is performed: QUESTION: IS BLANCO THE SAME GENE AS WHITE or TWO DIFFERENT GENES True breeding Blanco x true breeding white

7. 7 Mono and Di hybrid Crosses - review wwxWWwwxww (white)(red)(white)(white) F1WwF1ww (red)(white) -------------------------------------------- wwBBxWWbbwwbbxwwbb (white)(red)(white)(white) (Normal)(forked)(forked)(forked) F1WwBbwwbb (red eye)(white eye) (normal bristle)(forked bristle)

8. 8 The actual Cross Cross white (w) x blanco (b) WhiteBlanco Femalemale w/wxb/b

9. 9 White, Blanco, same gene? Precursor (white) Product (red pigment) Enzyme1 Gene1 White= Blanco yellow forked white blanco w w w w xw b w b (white eye)(white blanco eye) F1w w w b ????? In a self cross what percentage of flies would be red eyed and what percentage would be white eyed? All white

10. 10 White, Blanco, two genes? Precursor (white) Intermediate (blanco) Product (red pigment) Enzyme1Enzyme2 Gene1 White Gene2 Blanco yellow blanco forked white w w w w BBxWWbb (white eye)(blanco eye) F1w w WbB ????? In a self cross what percentage of flies would be red eyed and what percentage would be white eyed? 9red:7white

11. 11 The cross b Y w w What is the eye color of the w/Y males What is the eye-color of the w/b females? ? WHITE EYE IS X-LINKED What are genotypes and phenotypes of the cross wwxbY F1

12. 12 How many genes? The answer to the second question depends on whether the w and b mutations disrupt the same gene or different genes What if the w and b mutations disrupt the same gene? What if w and b mutations disrupt different genes?

13. 13 How many genes? The answer to the second question depends on whether the w and b mutations disrupt the same gene or different genes If the w/b females 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 b w b wB+ W+b b wB+ F1 wB+ W+b What if w and b mutations disrupt two genes? If the w/b females are white-eyed, we know that the white and blanco mutations disrupt one gene.

14. 14 If the females 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 ****Nomenclature**** If the researchers discover that blanco (b) and white (w) are mutations within the same gene. What are two mutations in the same gene called??? (allele) There is a problem. The same gene has two names. These are then renamed White becomes w 1 Blanco becomes w 2 or w b

15. 15 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 the w/b females 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

16. 16 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? Molecular basis of mutations

17. 17 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 ---CCCCACCCCCCCCCCCCCCCCCCCCCCCCC--- ---GGGGTGGGGGGGGGGGGGGGGGGGGGGGGG--- So at the molecular level, the w1/w2 white female fly would be depicted as: w1: ---CCCCCCCCCCCCCCCCCCCCCCCCCCTCCC--- ---GGGGGGGGGGGGGGGGGGGGGGGGGGAGGG--- w2: ---CCCCACCCCCCCCCCCCCCCCCCCCCCCCC--- ---GGGGTGGGGGGGGGGGGGGGGGGGGGGGGG---

18. 18 Genes Precursor white Intermediate white Product red Enzyme1Enzyme2 Gene A Gene B 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)

19. 19 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 ? Lets make things more complicated w1 and w2 disrupt one gene (A). w3 disrupts the second gene (B).

20. 20 Complementation analysis Genotypeeye color complementation phenotype Aw1/Aw2 Aw1/Bw3 Aw2/Bw3

21. 21 Suppose we isolate 5 curly wing mutations c1 c2 c3 c4 c5 We want to know how many genes are disrupted by these mutations and which mutations are in the same complementation group

22. 22 Complementation crosses We systematically perform crosses First we perform the cross c1/c1xc2/c2 F1c1/c2 are produced wing= flat or curly If they are flat, they disrupt -------- gene Then we perform c1/c1xc3/c3 F1c1/c3 wing=flat or curly You construct a complementation table + flat wing- is curly wing Mutation complementmutation don’t complement Different genessame gene c1c2c3c4c5 c1 c2 c3 c4 c5

23. 23 Intragenic recombination Up until this point, genes have been viewed as a linear array Of indivisible functional units on a chromosome The tenets of the model are 1Genes are fundamental units of function Parts of a gene cannot function 2Genes are fundamental units of mutation The gene changes as a whole from one form to another 3Genes are fundamental units of structure The gene is indivisible by recombination We will go through experiments that show that recombination does occur within a gene

24. 24 Recombination and genes If recombination occurs within a gene, how do you detect it? How do we detect recombination between two different genes? Drosophila: Two X-linked genes forked (f) is a recessive mutation that gives rise to forked bristles F=normal bristles f=forked bristles carnation is a recessive mutation that alters the normal bright Red-eyes to a dull red color C=normal red eyes c=dull carnation eyes Forked bristles carnation

25. 25 Recombination How do you detect recombination between these genes? fc F CxF C f cY FCFC fcfc FCFC Y The presence of individuals with recombinant phenotypes indicates that recombination has occurred between these two genes (X-linked)

26. 26 What about recombination within a gene Does recombination occur within a gene? Is there something special about a gene that makes it indivisible by recombination If recombination occurs within a gene how would you detect it? Recombination rates are a function of the distance between genes Greater the distance between genes, higher the recombination frequency For forked and carnation, Rf was approximately 10% These two genes are 240,000 bp apart To find out if recombination occurs within a gene we can look for recombination between two mutations within a gene f c F C f1 f2

27. 27 Recombination frequency Looking for recombination between two mutations within the same gene, we are dealing with extremely small rates of Recombination For example The Drosophila X chromosome is approximately 10 megabases (10,000,000 bps) The white gene (w) is about 1000 bp The white gene represents about 1/10,000th the length of the X-chromosome To look for recombination within a gene we perform the identical set of crosses used to look for recombination between genes w1 w2 w1 w2

28. 28 Recombination frequency To look for recombination within a gene we perform the identical set of crosses used to look for recombination between genes For example we isolate two independent mutations in the white gene w1 and w2 (How do we know that w1 and w2 are in the same gene?) w1 w2 w1 w2

29. 29 Intragenic recombination cross To detect rare recombinants between w1 and w2 We perform the following cross: w1/w2xW/Y w1 w2 W Y w1 w2 w1 w2

30. 30 Normal geneATG GGG GGG TTT CCC TTT AAA Mutant1ATG CGG GGG TTT CCC TTT AAA Mutant2ATG GGG GGG TTT CCC ATT AAA mut1 mut2 xnormal/Y Parental classes 1ATG CGG GGG TTT CCC TTT AAA 2ATG GGG GGG TTT CCC ATT AAA Recombinant classes 1ATG CGG GGG TTT CCC ATT AAA 4 ATG GGG GGG TTT CCC TTT AAA IC

31. 31 Definitions Intragenic recombination: Recombination occurring within a gene Intergenic recombination: Recombination occurring between genes Fine structure Analysis: Mapping a large number of mutations within a single gene through recombination CCCCCCCCCCCCCCCCCCCCTCCCC GGGGGGGGGGGGGGGGGGGGAGGGG CCACCCCCCCCCCCCCCCCCCCCCC GGTGGGGGGGGGGGGGGGGGGGGGG Analysis in the bacteriophage T4 reveals that recombination can occurs between single nucleotides