MATERI 7 DAN 8 SEX LINKAGE DAN CROSSING OVER

Gamet yang terbentuk dari individu dihibrid

Gamet yang terbentuk dari individu dihibrid A. Gen tidak berangkai B. Kedua Gen berangkai : Couple Phases, Kedudukan Sis Cara penulisan: (AB) ab C. Kedua Gen berangkai : Repulsion Phases, Kedudukan Trans Cara penulisan: (Ab) aB

Lalat Drosophila gen terangkai: Cu = gen untuk sayap normal cu = gen untuk sayap keriput Sr = gen untuk dada polos (normal) sr = gen untuk dada bergaris-garis

Cu Sr cu sr Cu sr cu Sr

Gen terangkai susunan sis

Gen terangkai susunan Trans

Rangkai Sempurna Susunan sis

Lalat jantan sayap keriput dada bergaris dikawinkan dengan lalat betina bersayap normal (sayap dan dadanya) homozigot
Betina

Cu Sr Cu Sr
F1

jantan

cu sr cu sr

Cu Sr cu sr
3:0;0;1

F2 :

Rangkai Sempurna Sususan Trans

Crossing Over of John Edwards Chromosomes

Linkage & Mapping

Linkage

Eukaryotic chromosomes are long ds DNA molecules Typical chromosome contains thousands of genes (loci) Linkage

loci located on the same chromosome linked loci tend to be transmitted as a unit

Linkage

Because they are a group of genes linked together, chromosomes are functionally linkage groups # of linkage groups = the # of types of chromosomes

Crossing over causes loci that are far apart on the same chromosome to sometimes independently assort

known as incomplete linkage

Crossing Over Produce Recombinant Phenotypes

Crossing over (meiotic recombination) Occurs during prophase I of meiosis at the bivalent stage

zygotene - pachytene - diplotene

Non-sister chromatids of homologous chromosomes exchange DNA segments

Linkage Prevents Independent Assortment

Figure 5.1

Crossing Over May Produce Recombinant Phenotypes

These are termed parental gametes

gametes with a combination of alleles NOT found in the original chromosomes as a result of meiotic recombination These are called nonparental or recombinant gametes

Figure 5.1

Example of Linkage

Bateson and Punnett conducted a cross in sweet pea involving two traits

Flower color and pollen shape

Dihybrid cross expected to give 9:3:3:1 phenotypic ratio of F2 phenotypes

Observed linkage Called it coupling

Example of Linkage
x Purple flowers, long pollen (PPLL) F1 offspring Red flowers, round pollen (ppll)

Purple flowers, long pollen (PpLl)

Self-fertilization
F2 offspring phenotypes
P NP NP P

Observed Observed Expected Expected Ratio Ratio number number

(9:3:3:1)
Purple flowers, long pollen Purple flowers, round pollen Red flowers, long pollen Red flowers, round pollen 296 19 27 85 15.6 1.0 1.4 4.5 240 80 80 27 9 3 3 1

Morgan Provided Evidence for the Linkage of Several X-linked Genes

The first direct evidence of linkage came from studies of Thomas Hunt Morgan Morgan investigated several traits that followed an X-linked pattern of inheritance

Body color Eye color Wing length

Linkage to a Particular Chromosome

F1 generation

y w m/y w m

y+ w+ m+ Y

Sex linkage of all traits places them all on X chromosome

y+w+ m+/ y w m

F1 generation contains wild-type females and yellow-bodied, white-eyed, miniature-winged males. ywm/Y

F2 generation

Females 439 208 1 5 7 0 178 365

Males 319 193 0 11 5 0 139 335

Total 758 401 1 16 12 0 317 700

Tan body, red eyes, normal wings Tan body, red eyes, miniature wings Tan body, white eyes, normal wings Tan body, white eyes, miniature wings Yellow body, red eyes, normal wings Yellow body, red eyes, miniature wings Yellow body, white eyes, normal wings Yellow body, white eyes, miniature wings

P Males

P Females

Morgan observed a much higher proportion of the combinations of traits found in the parental generation

Morgans explanation: All three genes are located on the X chromosome Therefore, they tend to be transmitted together as a unit
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Morgan Provided Evidence for the Linkage of Several X-linked Genes

However, Morgan still had to interpret two key observations

Why did the F2 generation have a significant number of nonparental combinations? Why was there a quantitative difference between the various nonparental combinations?

Reorganize Morgans data considering pairs of genes separately

Gray body, red eyes Yellow body, white eyes Gray body, white eyes Yellow body, red eyes Total Red eyes, normal wings White eyes, miniature wings 1,159 1,017 17 12 2,205 770 716

But this nonparental combination was rare

Red eyes, miniature wings White eyes, normal wings Total

401 318 2,205

It was fairly common to get this nonparental combination

Figure 5.4

These parental phenotypes are the most common offspring

These recombinant offspring are not uncommon

because the genes are far apart

Figure 5.4

These recombinant offspring are fairly uncommon

because the genes are very close together

These recombinant offspring are very unlikely 1 out of 2,205

it is product of single cross over probabilities

Incomplete Linkage

Linked loci that are sometimes separated by recombination are inherited in a pattern between linked and independently assorting

not always together not present in normal dihybrid ratios

The percentage of offspring with the loci linked vs those with the loci separated is a measure of the physical distance separating the loci on the chromosome

Alfred Sturtevants Analysis

An undergraduate in the laboratory of T. H. Morgan Constructed first genetic map in 1911 Sturtevant wrote:

In conversation with Morgan I suddenly realized that the variations in the length of linkage, already attributed by Morgan to differences in the spatial orientation of the genes, offered the possibility of determining sequences [of different genes] in the linear dimension of the chromosome. I went home and spent most of the night (to the neglect of my undergraduate homework) in producing the first chromosome map, which included the sex-linked genes, y, w, v, m, and r, in the order and approximately the relative spacing that they still appear on the standard maps.

Linkage and Genetic Maps

Estimating the relative distances between linked genes, based on the amount of recombination occuring between them allows us to generate genetic maps

If the genes are far apart many recombinant offspring If the genes are close very few recombinant offspring

Number of recombinant offspring X 100 Total number of offspring The units of distance are called map units (mu) They are also referred to as centiMorgans (cM) One map unit is equivalent to 1% recombination frequency

Map distance =

Linkage Analysis and Mapping

Genetic mapping experiments are typically accomplished by carrying out a testcross Example of a two-point mapping cross

Cross of two linked genes affecting bristle length and body color in fruit flies

s = short bristles + = normal bristles

e = ebony body color + = gray body color

One parent double recessive (homozygous recessive at both loci) s/s ; e/e Other parent is heterozygous at both loci (+/s ; +/e)

Chromosomes are the product of a crossover during meiosis in the heterozygous parent

Recombinant offspring are fewer in number than nonrecombinant offspring

Figure 5.9

5-47

Linkage Analysis and Mapping

The phenotype data are used to estimate the distance between the two loci

Number of recombinant offspring X 100 Map distance = Total number of offspring =

76 + 75 542 + 537 + 76 + 75

X 100

= 12.3 map units

Therefore, the s and e genes are 12.3 map units apart from each other along the same chromosome

Trihybrid Crosses

Data from trihybrid crosses can also yield information about map distance and gene order The following experiment outlines a common strategy for using trihybrid crosses to map genes In this example, we will consider fruit flies that differ in body color, eye color and wing shape

b = black body color b+ = grey body color pr = purple eye color pr+ = red eye color vg = vestigial wings vg+ = normal wings
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Step 1: Cross two true-breeding strains that differ at three loci.

Female is mutant for all three traits

Male is homozygous wildtype for all three traits

The goal in this step is to obtain F1 individuals that are heterozygous for all three genes
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Step 2: Perform a testcross by mating F1 female heterozygotes to homozygous recessive, male flies

During gametogenesis in the heterozygous female F1 flies, crossovers may produce new combinations of the 3 alleles
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Step 3: Collect data for the F2 generation

Phenotype Gray body, red eyes, normal wings Gray body, red eyes, vestigial wings Gray body, purple eyes, normal wings Gray body, purple eyes, vestigial wings Black body, red eyes, normal wings Black body, red eyes, vestigial wings Black body, purple eyes, normal wings
Number of Observed Offspring (males and females)

411 61 2 30 28 1 60

Black body, purple eyes, vestigial wings

412

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Analysis of the F2 generation flies will allow us to map the three genes

The three genes exist as two alleles each Therefore, there are 23 = 8 possible combinations of offspring If the genes assorted independently, all eight combinations would occur in equal proportions It is obvious that they are far from equal

In the offspring of crosses involving linked genes,

Parental phenotypes occur most frequently Double crossover phenotypes occur least frequently Single crossover phenotypes occur with intermediate frequency
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The combination of traits in the double crossover tells us which gene is in the middle A double crossover separates the gene in the middle from the other two genes at either end

In the double crossover categories, the recessive purple eye color is separated from the other two recessive alleles Thus, the gene for eye color lies between the genes for body color and wing shape
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Step 4: Calculate the map distance between pairs of genes To do this, one strategy is to group the data according to pairs of phenotypes resulting from non-crossovers, single crossovers, & double crossovers

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Phenotype

Number of Observed Offspring

30

Gray body, purple eyes, vestigial wings Black body, red eyes, normal wings Gray body, red eyes, vestigial wings Black body, purple eyes, normal wings Gray body, purple eyes, normal wings Black body, red eyes, vestigial wings

28

Single crossover between b and pr

30 + 28

= 0.058
1,005

61

60

Single crossover between pr and vg

61 + 60
= 0.120 1,005

Double crossover, between b and pr, and between pr and vg

1+2
= 0.003 1,005

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To determine the map distance between the genes, we need to consider both single and double crossovers

To calculate the distance between b and pr Map distance = (0.058 + 0.003) X 100 = 6.1 mu To calculate the distance between pr and vg Map distance = (0.120 + 0.003) X 100 = 12.3 mu To calculate the distance between b and vg

The double crossover frequency needs to be multiplied by two Because both crossovers are occurring between b and vg

Map distance = (0.058 + 0.120 + 2[0.003]) X 100 = 18.4 mu

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Step 5: Construct the map

Based on the map unit calculation the body color and wing shape genes are farthest apart The eye color gene is in the middle

The data is also consistent with the map being drawn as vg pr b (from left to right) In detailed genetic maps, the locations of genes are mapped relative to the centromere
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Alternatively, the distance between b and vg can be obtained by simply adding the map distances between b and pr, and between pr and vg Map distance = 6.1 + 12.3 = 18.4 mu

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Interference

The product rule allows us to predict the likelihood of a double crossover from the individual probabilities of each single crossover

P (double crossover) = P (single crossover X P (single crossover

between b and pr) between pr and vg)

= 0.061 X 0.123 = 0.0075

Based on a total of 1,005 offspring the expected number of

double crossover offspring is

= 1,005 X 0.0075 = 7.5

Interference

Therefore, we would expect seven or eight offspring to be produced as a result of a double crossover However, the observed number was only three!

Two with gray bodies, purple eyes, and normal sings One with black body, red eyes, and vestigial wings

This lower-than-expected value is due to a common genetic phenomenon, termed positive interference The first crossover decreases the probability that a second crossover will occur nearby
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Interference (I) is expressed as I = 1 C where C is the coefficient of coincidence

Observed number of double crossovers C= Expected number of double crossovers

C= 3 = 0.40

7.5

I = 1 C = 1 0.4 = 0.6 or 60% This means that 60% of the expected number of crossovers did not occur

Since I is positive, this interference is positive interference

Rarely, the outcome of a testcross yields a negative value for interference This suggests that a first crossover enhances the rate of a second crossover
The molecular mechanisms that cause interference are not completely understood However, most organisms regulate the number of crossovers so that very few occur per chromosome

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Somatic Cell Hybridization

Fusion of human cell and rodent cell Eventually, most human sequences lost

Lines carrying sub-sets of human chromosomes are compared to determine which express the given gene product

entire chromosomes, portions (arms) of chromosomes

Process of elimination determines on which chromosome (region of chromosome) a gene is located Hybrid Panel

have to have a detectible DNA sequence or protein

Other Mapping Techniques

Cytogenetic Mapping

Have a mutant phenotype Perform karyotype Observe altered chromosomes

aneuploidies translocations deletions

normal

Aberrant chromosome probably location of gene

translocation t(14;17)

Patau Syndrome - Trisomy 13 Karyotype: 47, 13+

Partial Chromosomal Deletions

cri-du-chat - 46, 5pFragile X mental retardation 46, XpMonosomy

loss of an entire chromosome lethal in all cases for autosomes XO Turners syndrome is only viable monosomy

Mapping Strategies

Deletion Mapping

Cross recessive heterozygote to deletion heterozygote lines appearance of recessive phenotype localizes recessive gene within deletion interval

Complementation

Determines if two phenotypes are caused by same mutant gene

Complementation Analysis

Complementation Analysis

Complementation Analysis of Eye Color Mutations

Complementation groups

white, cherry, coral, apricot, buff

W1, wch , wc , wa , wb Alleles of white gene

garnet ruby vermillion carnation

Complementation Analysis

Mapping with Unknown Phase of Linkage

When you have heterozygotes, but do not know what the phenotypes of the parents were.

12 possible diploid arrangements for 3 linked loci

bm pr

+ bm + pr

+ pr

+ bm + bm

brown midrib, virescent seedling, purple aleurone

Use Results of Cross to Determine Phase of Linkage

NCOs = parental chromosomes = phase

DCOs = fewest Do not directly detect middle locus

NCO Gives 3 Possibilities for Phase

12 possible diploid arrangements for 3 linked loci
bm pr

+ bm + pr

+ pr

+ bm + bm

DCO Gives Loci Order