Download Male Female vg + b + pr + vg b + pr + vg + b pr + vg b pr + vg + b + pr

Biology 423 Research Paper:
Genetics behind cloning of a human gene:
Topics due next week
1 page outlines due Oct. 29
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 Huntingtons syndrome or
Fibrodysplasia ossificans progressiva.
Collect papers
Define the problem you want to present
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 breathing
The 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 cells
Grant’s potassium pump message is expressed in lung cells
Treatment 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 paragraphs
First sentence is a topic sentence
Last sentence is a concluding sentence
active voice
don’t try to sound academic
define your terms
if referring to a figure, define the figure but do not duplicate the figure legen
Maintain the same tense, either past or present.
Make and 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 order
Smith 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 cross
Body color; yellow vs wild type
Bristles: 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
Female
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 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
To map a human genetic trait:
Look for association between mapped
markers and a trait of interest
Markers can be traits, proteins or DNA sequences
Anything that is polymorphic can be mapped
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
RFLP markers linked to trait
RFLP polymorphisms reveal genetic differences
2. Separate DNA fragments by
size on an agarose gel
3. Hybridize to single copy
radioactive probe- Southern Blot
1. Cut genomic DNA with
Restriction enzymes
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, we estimate map distance as 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).
In our case:
L = 6.1
Log of L or LOD = 0.8
Maximum likelihood odds of linkage; Change estimated linkage
Distance p(.1) to get the best LOD score for the data.
Calculate Lod score
• Odds of linkage
– Probability gene and marker are linked at
a certain map distance / Probability they
are unlinked.
– Calculate maximum odds for data.
Predicted linkage distance gives best odds
– Add up log of odds for many families to
get more data
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 accepted
Linkage 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 LOD will be
Negative.
Therefore, as you add more families the
LOD will only increase if the data of the majority
of families supports linkage.
Huntington’s Disease
A neurodegenerative condition that results in degeneration of
basal ganglia late in life.
Autosomal Dominant
Fully penetrant
Affects 1 in 10,000 individuals
One of the first genes identified by map based cloning
Gusella et al 1984. Science 225, 1320-1326
100’s of DNA markers mapped onto each chromosome –
high density linkage map.
the relative location of 100s of polymorphic DNA markers
on chromosomes can be mapped using mapping panels.
First step. Find approximate chromosomal location of gene using
few large families or many small families.
Use LOD score to determine if markers are linked to gene in
human families. The LOD score allows you to compare families
in which marker and gene are either in repulsion or in coupling.
Gusella et al 1984. Science 225, 1320-1326
DNA was collected from family members
Many sequences known to be polymorphic in humans
Were tested to find sequences that are linked to disease.
i.e. find polymorphic alleles that cosegregate with disease allele.
RFLPs were tested. Restriction Fragment Length Polymorphisms
One locus with a HindIII polymorphism was linked
in two large families tested.
HindIII polymorphism is closely linked to disease
Marker G8 from a randomly chosen phage clone with
a 17.6 kb human DNA insert.
Allele C is always found in affected
individuals with one exception
Marker G8 is linked to Huntingtons disease at a distance of 2 cM
With a LOD score of 12.1
Some unaffected individuals also have allele C
but in these cases it is from an unaffected parent
Physical map of region that contains Huntingtons gene
Huntington Disease Collaborative Research Group 1993
75 families from all over the world tested for closer markers
500 kb region between D4S180 and D4S182 – no recombinations
Once a small interval has been defined,
how to find the correct gene?
List of genes predicted from sequence.
Examine mRNA expression patterns to focus on
genes expressed in tissues affected by disease.
Compare alleles of candidate genes between
healthy and affected individuals.
Exon trap used to identify expressed genes
in Huntingtons interval
Bucker et al. 1991 PNAS 88, 4005-4009
Screened sequences from clones representing contig,
4 transcripts found.
IT15 encoded a gene that was different in healthy
family members from affected family members.
Expressed in many tissues including brain
Structure of Huntingtin protein
Polyglutamine repeats (CAG codon) are
expanded in diseased individuals
Sawa 2001 J. Mol. Med. 79, 375-381.
Once the gene is available,
new studies can be initiated to understand disease
Mouse model: transgenic mouse with expanded polyglutamine repeats.
Heterozygote has neurodegenerative disease similar to
Huntington’s.
Develop abnormal deposits of huntingtin fragments in inclusion
bodies of neurons.
Express huntingtin protein in cultured nerve cells
Mutant fragments accumulate in clumps and cells die
Normal protein also fragmented but clumps do not develop.
May be clumps are toxic or
Maybe fragments with longer polyglutamine repeats are toxic.
Possible toxic effects of huntingtin leading
to nerve cell death
Huntingtin binds some transcription factors.
Mutant Htt also binds
May take transcription factors out of action.
Mutant but not normal Htt binds p53 transcription factor,
p53 is a regulator of cell death
As a result,Ca2+ flow altered in mitochondria, Cell death.
Mutant also binds a histone deacetlyase.
Modification of gene expression.
Normal Huntingtin may protect nerve cells from death by performing
essential functions.
Normal Htt is part of a complex that transports a hormone,
Brain derived neurotrophic factor
along axons of medium spiny neurons.
These are first to degenerate in Huntingtons Disease.
Mutant Htt cannot perform transport function.
But HD is dominant .
It may be toxic or heterozygotes may have insufficient
functional protein for normal development.
Haploinsufficient.
Possible functions of Huntingtin leading to disease
Although the function of Huntingtin is still not known,
treatments are being tested to reduce the symptoms
Caspase blockers
Anti-apoptosis