1. Recombination and Mapping (cont’d)

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.
1) Genes far apart, double, triple etc crossovers affect MU
2) Species specific differences
Humans 1MU is ~ 1 million bp;
Yeast 1MU is ~ 5000 bp
3) Sex specific differences
4) Chromosome Position effects

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. Recombination hot spots
geneA
geneB
geneC
geneD
geneE A B C d e D E a b c
hotspots of recombination and recombination deserts*****

5. Chromosome Position specific effects on recombination
Cen
Tel
Recomb
Drosophila X Fw Wy Fa Pa
2MU
0.5x106 bp
High Recombination
1x106 bp
Low recombination
Fa and Pa are 2MU apart -low recombination freq
Fw and Wy are 2MU apart- high recombination freq
Real distance
Fa-Pa = 1x106 bp
Fw-Wy = 0.5x106 bp
Fw-Wy are closer together along the DNA but because of higher recombination rate they appear to be farther apart

6. Recombination hot spots
Recombination rate along human chromosome 12

7. Determinants of meiotic recombination
In yeast:
Occur preferentially in “open” promoters of genes
Suppressed in “closed” centromeres, telomeres
In humans/mouse:
More recombination in gene dense regions
More often in intergenic regions over genes
Rare in closed heterochromatin
13 nucleotide motif CCNCCNTNNCCNC accounts for >40% hotspots. Mutation in this sequence reduces recombination

8. J. C. Roach et al., Science 328, 636-639 (2010)
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
dad
mom
J. C. Roach et al., Science 328, (2010)

9. xxxxxxxxx

10. Intragenic recombination
Up until this point, genes have been viewed as a linear array of indivisible functional units on a chromosome A B C
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

11. 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

12. How do you detect recombination between these genes?f c
F C x F C
f c Y
(X-linked)
F C
f c
F C Y FC Y FC WT WT
400
parental fc WT
Forked carnation
400 Fc WT
carnation 50
recom fC WT
forked 50
100/900
The presence of individuals with recombinant phenotypes
indicates that recombination has occurred between these two genes
Recombination frequency is ~10%

13. 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

14. 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

15. 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

16. Intragenic recombination crossw1 w2
To detect rare recombinants between w1 and w2
We perform the following cross:
w1/w2 x W/Y w1 w2 W Y W Y w1
W/w1 Red
w1/Y White
parental w2
W/w2 Red
w2/Y White w2 w1
W/w1,2 Red
w1,2/Y White
recom W
W/W Red
W/Y Red

17. 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

18. Normal gene ATG GGG TTT CCC TTT AAA
Mutant1 ATG CGG TTT CCC TTT AAA
Mutant2 ATG GGG TTT CCC ATT AAA
mut1 x normal
mut Y
Parental classes
1 ATG CGG TTT CCC TTT AAA
2 ATG GGG TTT CCC ATT AAA
Recombinant classes
3 ATG CGG TTT CCC ATT AAA
4 ATG GGG TTT CCC TTT AAA IC

19. CCCCCCCCCCCCCCCCCCCCTCCCC GGGGGGGGGGGGGGGGGGGGAGGGG
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

21. The Big Picture: an outline of the concepts covered to date
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 B b

22. 4. Exceptional patterns of chromosome segregation
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 (self cross1:2:1)
Co-dominance (self cross1:2:1)
Lethal alleles (self cross 1:2)
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

23. linkage
A b a b
a B x a b >1:>1:<1:<1
>25%:>25%:<25%:<25%
a b c d e f g
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 MU
What is the recombination freq between a and f?
Is it 80%, less than 80%, more than 80%

24. a b c d e f g 10 20 15 5
a f
af P
aF R
Af R
AF P
a f
A F
A F
Recombinant/total = 2/4=50%
Maximum freq is 50%

25. 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

26. 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/protein
chromosome
yellow
white
Shaven body
Forked
bristle
blanco
Many genes
Genes on
DNA
white1
white2