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.

-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

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

Sex specific differences: human chromosome 12.

Recombination hot spots

5

gen

eA

gen

eB

gen

eC

gen

eD

gen

eE

D Ea b c

A B C d e

-hotspots of recombination and recombination deserts*****

6

Chromosome Position specific effects on recombination

Fa and Pa are 2MU apart -low recombination freqFw and Wy are 2MU apart- high recombination freq

Real distanceFa-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

Cen Tel

RecombFreq

Drosophila X

Fw Wy Fa Pa

2MU 2MU

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?

Recombination hot spots

Recombination rate along chromosome 12

xxxxxxxxx

9

10

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

1 Genes are fundamental units of functionParts of a gene cannot function

2 Genes are fundamental units of mutationThe gene changes as a whole from one form to another

3 Genes are fundamental units of structureThe 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 bristlesf=forked bristles

carnation is a recessive mutation that alters the normal bright Red-eyes to a dull red color

C=normal red eyesc=dull carnation eyes?

Fork

ed

bri

stl

es

carn

ati

on

12

Recombination

How do you detect recombination between these genes?

f c

F C x F Cf c Y

F C

f c

F C

Y

The presence of individuals with recombinant phenotypesindicates that recombination has occurred between these two genesRecombination frequency is ~10%

(X-linked)

parental

recom

FC Y

FC

fc

Fc

fC

WT

WT

WT

WT

WT

Forked carnation

carnation

forked

400

400

50

50

100/900

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 exampleThe Drosophila X chromosome is approximately 10 megabases (10,000,000 bps)The white gene (w) is about 1000 bpThe 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 cross

To detect rare recombinants between w1 and w2We perform the following cross:

w1/w2 x W/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

w2w1

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)

8/10,0000.08 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 GGG TTT CCC TTT AAA

Mutant1 ATG CGG GGG TTT CCC TTT AAA

Mutant2 ATG GGG GGG TTT CCC ATT AAA

mut1 mut2 x normal/Y

Parental classes

1 ATG CGG GGG TTT CCC TTT AAA

2 ATG GGG GGG TTT CCC ATT AAA

Recombinant classes

3 ATG CGG GGG TTT CCC ATT AAA

4 ATG GGG GGG TTT CCC TTT AAA

IC

19

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

CCCCCCCCCCCCCCCCCCCCTCCCCGGGGGGGGGGGGGGGGGGGGAGGGG

CCACCCCCCCCCCCCCCCCCCCCCCGGTGGGGGGGGGGGGGGGGGGGGGG

Analysis in the bacteriophage T4 reveals that recombination can occurs between single nucleotides

20

21

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

aA

a

B

bb

B

22

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 lawsat the level of the gene, but Phenotype ratios are modified

23

a b c d e f g10 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%

24

a b c d e f g10 20 20 15

15 5

a f

a f

A F

A F

af PaF RAf RAF P

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

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 color chr1

Flower color chr1

Pod shape chr4

Flower position chr4

Stem length chr4

Pod color chr5

Seed shape chr7

27

Ratios

A Aa x a 3:1

A aa x a 1:1

A b A ba B x a B 9:3:3:1

A b a ba B x a b 1:1:1:1

28

Chromosomes, genes, alleles, proteins, phenotypes

Each chromosome in G1 has one DNA moleculeEach 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

wh

iteyellow

Sh

aven

bod

y

Fork

ed

b

ristl

e

bla

nco

white1

Many genes

Genes on DNA

white2

chromosome