Biology 423 Research Paper:Genetics behind cloning of a human gene:

1 - 2 page outlines due Nov. 6

Writing a scientific paper:

Choose a topic: Pick a genetic disease for which the responsible gene has been cloned. You can find lists of these diseases at the following Yahoo site: http://dir.yahoo.com/Health/Diseases_and_Conditions/Genetic_Disorders/The OMIM database is also a useful place to get information to start.

Please do not choose Cystic Fibrosis or Fibrodysplasia ossificans progressiva.

Collect papers:Look for genetics studies, family studiesas well as physiology, biochemistry and cell biology

Make a title – active statements work well eg. Grant’s disease is caused by a defective potassium pump

Make an outline:Define a title for each section – even each paragraph. Use active statements again.

eg. Grant’s disease is a genetic disease that affects breathingThe Grant’s disease mutation is on Chromosome 7

A mutation in a potassium pump is linked to Grant’s disease(The two above will be the main sections)

Expression of wild type potassium pump reverts Grant’s disease effects in cultured cellsGrant’s potassium pump transcripts are expressed in lung cellsTreatment of patients with potassium pump blockers has no effect on progress of disease. Microarray analysis suggests potential treatment.

These can be used as section titles or as topic sentences (see below)

Choose figures and make them. You can copy figures from some of the papers you read. If so, cite them properly eg (from Smith et al., 1987). The figure with its figure legend should be comprehensible without reading the text.

Write paragraphsFirst sentence is a topic sentence Last sentence is a concluding sentence active voicedon’t try to sound academicdefine your terms, (I am not a medical doctor)if referring to a figure, define the figure but do not duplicate the figure legend. Maintain the same tense, either past or present.

Make an abstract to put at the beginning of the paper summarizing the experiments you will describe, the results and the conclusions.

Citations: When you discuss published work, cite the paper. Do the citation in the first sentence in which the study is mentioned.Eg. Seven large families with a high incidence of cystic fibrosis were surveyed for DNA markers linked to the disease (Smith et al., 1987).

References: at the end of the section: in alphabetical orderSmith J, Jones, P.A. and White, K. 1987 Family studies map cystic fibrosis to Chromosome 7 Genetics 130: 147-156. Use the journal “Cell” as an example of how to format the paper, the citations and the references.

There are several nice reference managers available. We use EndNote for making bibliographies and storing references.

Mapping genes by recombination frequency

Test cross to monitor recombination between different genes

Frequency of recombination is directly related to distance between genes (loci) on chromosome

Three point cross

Drosophila, a model organism for genetics

Traits for our three point crossBody color; yellow vs wild typeBristles: forked vs straight (wild type)Crossveins: crossveinless vs wild type

Fig. 5.12

Test cross

vg b pr / vg+ b+ r+ X vg b pr / vg b pr

Punnet square:

Male Femalevg+b+pr+ vg b+pr+ vg+b pr+ vg b pr+ vg+b+ pr vg b+pr vg+b pr vg b pr

Vg b pr vg+b+pr+ vg b+pr+ vg+b pr+ vg b pr+ vg+b+ pr vg b+pr vg+b pr vg b pr

1:1:1:1:1:1:1:1 ratio of phenotypes if genes are not linked

If genes are linked, parental combinations of alleles are overrepresented in progeny

Fig. 5.12

3 genes, which is in the middle?

Fig. 5.13

Calculate distance between pairs: vg to pr: add up all classes with a recombination event between vg+ and pr or vg and pr+

252 + 241 + 13 + 9 = 525

Divide by total number of chromosomes scored:

525/4197 X 100 cM = 12.5

Calculate distance between pairs: pr to b: add up all classes with a recombination event between pr+ and b or pr and b+

131 + 118 + 13 + 9 = 271

Divide by total number of chromosomes scored:

271/4197 X 100 cM = 6.4

The distance between vg and b is the sum of the distance between vg-pr and pr-b 12.3 + 6.4 = 17.7

Fig. 5.12

Fig. 5.15

How do we map genes in humans?

Relative association of markers:Allelic variants will co-segregate if the genes are closely linked on a chromosome.

Map distances depend on frequency of recombination

Markers can be traits, proteins or DNA sequences

Anything that is polymorphic can be mapped

To map a human genetic trait:Look for association between mapped markers and a trait of interest

We can translate map position into DNA sequence by determining the linkage between DNA-based markers and traits.

Pedigree Analysis: symbols

Screen family members for DNA markers linked to trait

Screen family members for RFLP markers linked to trait

RFLP polymorphisms reveal genetic differences

1. Cut genomic DNA with Restriction enzymes

2. Separate DNA fragments by size on an agarose gel

3. Hybridize to single copy radioactive probe- Southern Blot

HindIII polymorphism is closely linked to disease

Marker G8 from a randomly chosen phage clone with a 17.6 kb human DNA insert.

N D D N N D D N N N

Test degree of linkage: odds of linkage

Data looks like M1 is linked to SF. Mother has two M1 alleles.Her chromosome is uninformative, like a test-cross. Father has two different M alleles. Recombination of his alleles can be seen in this pedigree.

In this family, there are 8 informative chromosomes.1 has a recombination event.therefore, a rough estimate of map distance is 1/8X 100 cM = 12.5%

Odds of Linkage is

(Probability gene and marker are linked at a certain map distance) divided by (Probability they are unlinked).

Maximum likelihood odds of linkage; Change estimated linkage distance (θ) to get the best LOD score for the data.

example

In our case: best odds of linkage value for this pedigree is L(.10) = 6.3

Log of L or LOD = 0.8

Maximum LOD score = Zmax = 0.8

• θ = recombinant fraction

• L(θ) = odds of linkage at 0 > θ < 0.5

• L(0.5) = odds of independent assortment

• Log[L(θ)/L(0.5)] = log-of-odds ratio = LOD = Z

• LOD scores > 3 indicate linkage

• LOD scores < -2 indicate non-linkage

LOD nomenclature

LOD score is used to determine if two traits are linkedin human pedigrees

θ 0.001 0.01 0.05 0.1 0.2 0.3 0.4

Z -6.0 -3.0 -1.1 -0.4 0.1 0.2 0.1

θ 0.05 0.1 0.15 0.2 0.25 0.3 04

Σ(Z) 28.2 31.2 30.4 27.8 24.0 19.4 9.0

Example linkage of marker and trait

Zmax = maximum likelihood score (MLS)

To achieve significant LOD score:

Combine odds of linkage for many families:

p1(L)/p1(NL) x p2(L)/p2(NL) xp3(L)/p3(NL)

In practice we combine the log of odds:

LOD1 + LOD2 + LOD3.

Continue until LOD > 3.0 before linkage is acceptedLinkage distance is based on the linkage distance that gives the maximum value for the data.

If genes and markers are unlinked, the p(L)/p(NL) will be <1.0 in some families and the final LOD score will be negative (<0).

Therefore, as you add more families the LOD score will only increase if the data of the majority of families supports linkage.

Genetic distance between two genes is measured by the recombination frequency of alleles of those genes.

If genes are closely linked, alleles for those traits will not become recombined as often as if the genes are more distant on a chromosome.

Genome maps can be made using combinations of data from relative recombination distances of many loci.

Human genetic linkage between two traits or two genes is measured using Log scores.

Log scores greater than 3.0 (1/1000) chance of non-linkage are the minimum values accepted as evidence of linkage in family studies.

Summary