1. 1 Recombination and Mapping (cont’d)

2. 2 Factors affecting MU In most cases the order of genes revealed by mapping techniques correspond to the order of genes determined by sequencing. In contrast, actual physical distance between genes does not show direct correspondence to map units. Gene order, but not gene distance, is usually consistent between genetic and physical maps. -for genes far apart, double, triple etc crossovers affect MU -Species specific differences Humans 1MU is ~ 1 million bp; Yeast 1MU is ~ 5000 bp -Extreme example: In Drosophila males, there is no recombination during meiosis

3. Sex specific Differences 3 -Sex specific differences For example markers D12s7 and Pah males Rf= 9% females Rf= 22% Extreme example: In Drosophila males, there is no recombination during meiosis

4. 4 Sex specific differences: human chromosome 12.

5. Recombination hot spots 5 geneA geneB geneC geneD geneE DEabc ABCde -hotspots of recombination and recombination deserts*****

6. 6 Chromosome Position specific effects on recombination Fa and Pa are 2MU apart -low recombination freq Fw and Wy are 2MU apart- high recombination freq Real distance Fa-Pa = 1x10 6 bp Fw-Wy = 0.5x10 6 bp Fw-Wy are closer together along the DNA but because of higher recombination rate they appear to be farther apart Cen Tel Recomb Freq Drosophila X Fw Wy FaPa 2MU

7. 7 One major reason for this is that the recombination rates are not equal through the length of the chromosome. 2m.u lies between Pa and Fa- these two genes are located near the telomere 2m.u lie between Fw and Wy- these two genes are located in the middle of the chromosome What can you conclude about the physical distance between these two sets of genes?

8. Recombination hot spots Recombination rate along chromosome 12

9. 9 Sequencing the whole genome of a family: On average ~100 crossovers in maternal genome On average ~60 crossovers in paternal genome Recombination is 1.7x more frequent in females than males ~100 out of ~160 Crossovers were at hotspots of recombination J. C. Roach et al., Science 328, 636-639 (2010) dadmom

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11. 11 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

12. 12 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 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

13. 13 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 Recombination frequency is ~10% (X-linked) parental recom FC Y fc Fc fC WT Forked carnation carnation forked 400 50 100/900

14. 14 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 the same gene f c F C f1 f2

15. 15 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

16. 16 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

17. 17 Intragenic recombination cross To detect rare recombinants between w1 and w2 We perform the following cross: w1/w2xW/Y w1 w2 W Y Y W/w1 Red W w1 w2 W w1/Y White w2/Y White w1,2/Y White W/Y Red w1 w2 w1 w2 parental recom W/w2 Red W/w1,2 Red W/W Red w2 w1

18. 18 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 white and have 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

19. 19 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 3ATG CGG GGG TTT CCC ATT AAA 4 ATG GGG GGG TTT CCC TTT AAA IC

20. 20 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

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22. 22 Mitotic recombination Although not as frequent, recombination between homologous chromosomes occasionally occurs in mitosis as well as meiosis. This was first discovered in Drosophila by Kurt Stern and now has important implications for the origins of some human cancers Stern made the following cross: ｷ y = yellow body ｷ sn = singed bristles ｷ y+ = normal body ｷ sn+ = normal bristles The F1 females These should be phenotypically normal

23. 23 Mitotic recombination Although not as frequent, recombination between homologous chromosomes occurs occasionally in mitosis as well as meiosis. This was first discovered in Drosophila by Kurt Stern and now has important implications for the origins of some human cancers Stern made the following cross: ｷ y = yellow body color ｷ sn = singed bristles ｷ y+ = normal body color ｷ sn+ = normal bristles y+ snxy sn+ y+ sny sn+ The F1 females y+ sn y sn+ These should be phenotypically normal

24. 24 Mitotic recombination However, some females in a background of normal bristle and normal body color had sectors of singed bristle, normal colored tissue next to sectors of yellow body color normal bristle length. Because these sectors were adjacent to one another, Stern thought that the two spots must be reciprocal products of the same event. That event may be a rare crossover between homologs during the mitotic divisions. y+ sn y sn+

25. 25 A normal mitotic division would occur as follows: y+ sn y sn+ Replication Segregation y+ sny sn+ y+ sn y sn+ y+ sny sn+ y+ sny sn+ Genotypically identical Daughter cells

26. 26 If you have recombination in mitosis Replication y+ sny sn+ y+ sny sn+ Genotypically different Daughter cells Segregation

27. 27 If you have recombination in mitosis Replication y+ sn y sn+ Genotypically different Daughter cells Segregation y+ sn y sn+ y+ sn y sn+ y+ sn y sn+ y+ sn y sn+ y+ sn y sn+

28. 28 Mitotic recombination This only happens in somatic cells in your body It is therefore not passed on to your progeny. So Why is this important?

29. 29 Rb While mitotic recombination is rare, it does occur. It can convert a heterozygous cell into a homozygous mutant cell and a wild-type. Formation of homozygosity in cells can be carcinogenic if a mutated gene becomes homozygous in somatic cells!!! Retinoblastoma (Rb) is a human tumor that sometimes results from a mitotic recombination event. Rb is a childhood cancer of the eye It occurs from birth to 4 years of age If discovered early enough, treatment is 90% effective

30. 30 Rb There are two forms of Rb: Hereditary and sporadic Hereditary Rb: patients typically develop multiple eye tumors involving both eyes. These tumors develop at an early age. Siblings often develop the same sort of tumors. Sporadic Rb (60% of the cases): The development of the eye tumor is a spontaneous event in the patient with no history of the disease. Tumors develop only in one eye. Occurs later than hereditary Rb

31. 31 Sporadic Retinoblastoma Rb+/Rb+ (phenotypically normal) First point mutation Rb+/Rb- cell (phenotypically normal) Second mutation Rb-/Rb- cell (eye tumor) Hereditary Retinoblastoma Rb+/Rb- (phenotypically normal) Mutation Rb-/Rb- cell (eye tumor)

32. 32 Knudson proposed the following model to explain both forms of Rb: In Knudson's model of hereditary Rb, instead of a mutation producing cells homozygous for the Rb gene, a mitotic recombination event could also result in homozygosity for the Rb gene Rb+/- Rb+/+ Rb+/- - + Second Point mutation Chromosome loss Mitotic recombination - - -- - Inherited Non-hereditary

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34. 34 The Big Picture: an outline of the concepts covered to date 1.Genes are physical units of hereditary that carry information from one generation to the next 2. Mendel elucidated the following principles regarding the inheritance patterns of genes A. Each diploid individual contains two copies of a given gene B. Each Gene can have different forms called alleles. There are two alleles in a diploid individual The form that is expressed phenotypically in the heterozygote is known as the dominant allele. It is an operational definition C. These copies (alleles) segregate from one another to form gametes. There is a single copy of each gene in a gamete (one allele of a gene in a gamete) D. Different genes assort independently from one another during gamete formation (unless they are on the same chromosome and are linked) 3. The inheritance pattern of genes parallels the behavior of chromosomes at meiosis. This generated the hypothesis that genes reside on chromosomes OR A a A a B b b B

35. 35 The Big Picture 4. Exceptional patterns of chromosome segregation The X/X and X/Y sex chromosomal system produces exceptional segregation patterns because males contain only one copy of X-linked genes Non-disjunction: homologous chromosomes migrate to the same pole during meiosis 5. Exceptional expression (phenotype) patterns: Incomplete dominance, Co-dominance, Lethal alleles 6. Genes that reside close to one another on the same chromosome do not assort independently- linkage +++++exceptions to Mendellian laws at the level of independent assortment of two genes+++++ 7. Occasionally recombination occurs between these linked genes. The higher the frequency of recombination between any two genes, the greater the distance between them. Recombination frequencies serve as a useful method of mapping genes along a chromosome. No exceptions to Mendellian laws at the level of the gene, but Phenotype ratios are modified

36. 36 a b c d e f g 10 20 20 15 15 5 This map means that there is a 20% recombination frequency between the genes b and c and a 5% recombination frequency between the genes f and g Genes very far apart on the same chromosome will appear to assort independently How many map units between a and f? a-f = 80 cM What is the recombination freq between a and f? Is it 80%, less than 80%, more than 80%

37. 37 a bc d e f g 10 20 15 5 afaf afaf AFAF AFAF afP aFR AfR AFP Recombinant/total = 2/4=50% Maximum freq is 50%

38. 38 The largest distance that can be measured by this technique is 50MU. 50% also indicates NO LINKAGE If two genes are very far apart on the same chromosome, use markers between these genes to more accurately map the genes Therefore when you obtain a recombination frequency of 50% this means that either: the genes are on two different chromosomes OR very far apart on the same chromosome

39. 39 Mendel studied 7 traits that assorted independently. The only explanation for this behavior is that the genes controlling these traits are located on different chromosomes. True False Seed colorchr1 Flower colorchr1 Pod shapechr4 Flower positionchr4 Stem lengthchr4 Pod colorchr5 Seed shapechr7

40. 40 Ratios AA axa3:1 Aa axa1:1 A bA b a Bxa B9:3:3:1 A ba b a Bxa b1:1:1:1

41. 41 Chromosomes, genes, alleles, proteins, phenotypes Each chromosome in G1 has one DNA molecule Each chromosome in G2 has two DNA molecules (2 sister chromatids) Each chromosome has many genes A gene has many forms- alleles - two alleles in a diploid Each allele produces a protein that give rise to a phenotype Different alleles are caused by different changes in the same gene white yellow Shaven body Forked bristle blanco white1 Many genes Genes on DNA white2 chromosome