w1 w2 w3 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 chromosome Forked bristle Many genes blanco yellow Shaven body white Genes on DNA w1 w2 w3 Mutations in a gene

Mutant1+ citruline=arginine Mutant2+citruline=mutant 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. Ornithine Citruline Arginine Enzyme1 Enzyme2 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!

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

White and Blanco QUESTION Are the two independently isolated mutations disrupting the same or different genes. Precursor (white) Intermediate Product (red pigment) Enzyme1 Enzyme2 Gene1 White Gene2 Blanco Blanco=White???? OR

White gene Mapping genes takes lots of crosses and is time consuming 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 If the two mutations map to different regions of the X chromosome 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!

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

Cross White Blanco Female male w/w x b/b The actual Cross white (w) x blanco (b) White Blanco Female male w/w x b/b white and blanco are on X-chromosome

(white eye) (blanco eye) White, Blanco, two genes? Precursor (white) Intermediate (blanco) Product (red pigment) Enzyme1 Enzyme2 Gene1 White Gene2 Blanco forked blanco yellow white wwwwBB x WWbb (white eye) (blanco eye) F1 Www Bb ????? In a self cross what percentage of flies would be red eyed and what percentage would be white eyed? 9red:7white

What are genotypes and phenotypes of the cross ww x bY WHITE EYE IS X-LINKED What are genotypes and phenotypes of the cross ww x bY b Y w F1 w What is the eye color of the w/Y males What is the eye-color of the w/b females? ?

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?

****Nomenclature**** Single gene 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. 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 w1 Blanco becomes w2 or wb

Two genes 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 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)

Two genes Precursor white Intermediate Product red Enzyme1 Enzyme2 If the 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 Precursor white Intermediate Product red Enzyme1 Enzyme2 gene1 gene2 blanco w B+ W+ b yellow singed Shaven body forked white blanco

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?

Mutant genes Nucleotide sequence of the normal W gene: ---CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC--- ---GGGGGGGGGGGGGGGGGGGGGGGGGGGGGG--- The sequence of the w1 mutation of gene W - The sequence of the w2 mutation of gene W So at the molecular level, the w1/w2 white female fly would be depicted as: w1: w2:

Genes Precursor white Intermediate Product red Enzyme1 Enzyme2 Gene A Lets make things more complicated. w1 and w2 disrupt one gene (geneA). w3 disrupt a second gene (geneB) Precursor white Intermediate Product red Enzyme1 Enzyme2 Gene A Gene B 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

Complementation analysis Genotype eye color complementation phenotype Aw1/Aw2 N Aw1/Bw3 Y Aw2/Bw3 Y

Suppose we isolate 5 curly wing mutations We want to know how many genes are disrupted in these mutations and which mutations are in the same complementation group

Complementation crosses We systematically perform crosses First we perform the cross c1/c1 x c2/c2 F1 c1/c2 are produced wing= flat or curly If they are flat, they disrupt -------- gene Then we perform c1/c1 x c3/c3 F1 c1/c3 wing=flat or curly You construct a complementation table + flat wing - is curly wing Mutation complement mutation don’t complement Different genes same gene c1 c2 c3 c4 c5 c1 c2 c3 c4 c5

The pathway Precursor curly Intermediate curly Product flat Enzyme1 Enzyme2 Gene1 (allele c1, c2, c5) Gene2 (allele c3, c4)

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 Genes are fundamental units of function Parts of a gene cannot function Genes are fundamental units of mutation The gene changes as a whole from one form to another Genes 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

Recombination and genes If recombination occurs within a gene, how do you detect it?

Recombination and genes If recombination occurs within a gene, how do you detect it? How do we detect recombination between two different genes? carnation Forked bristles

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 carnation Forked bristles

Recombination f c F C x F C f c Y F C f c Y How do you detect recombination between these genes? f c F C x F C f c Y F C f c Y (X-linked) The presence of individuals with recombinant phenotypes indicates that recombination has occurred between these two genes

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 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 w1 w2 w2

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

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

The result The cross produced the following results 10,000 males obtained 9996 were white eyed 4 were red eyed Map distance = # recombinants/total progeny 4+4/10,000 (why 4+4?) because 4 recombinants were ehite and hhave to be added to the 4 red to get total recombinants) 8/10,000 0.08 MU or cM The results demonstrate That the gene is divisible by recombination Mutations in a gene occur in different positions within that gene