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New Homework Assignment

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• Exam 2 Answer Key • Final Grade Info • Regrade Requests

3. The Human Genome in the Post-Genomic Era
What we did (and didn’t) learn from the human genome project.
How does the human genome compare to those of
other species?
What are some of the mechanisms that contribute
to biological complexity?
Epigenetics: Another way of looking at
the genome.
Maybe Lamarck was (partially) right.

4. The Human Genome Project
Began in Completed in 2003
There are roughly 3 billion base pairs in the (haploid) human genome
There are roughly 25, 000 genes in the human genome.
How do these values compare to those of other species?

5. Who has the largest genome? the greatest number of genes?
human
flu virus
E. coli (bacterium)
chicken
fruit fly
grapes

6. Gene Number

7. One measure of
genome size

8. Where does all the complexity come from?
Only ~1.5% of the
~3 billion bases in the human genome directly encode protein.

9. One Gene, Many Gene Products: 1) Alternative Splicing

10. One Gene, Many Gene Products: 2) Alternative Promotors
hepatocyte nuclear factors

11. One Gene, Many Gene Products: 3) RNA Editing
Amino acid substitution
Splice site alteration
Alteration of secondary structure

12. RNA Editing Adenosine Deaminase Acting on RNA Robert Reenan
Brown University
Adenosine Deaminase Acting on RNA
(ADAR)
ADAR Catalyzes the deamination of Adenosine to Inosine
The ribosome reads inosine as guanine
Most ADAR targets are transcipts involved in rapid neuronal transmission

13. Searching for “Disease Genes”
“Disease Gene” is a misnomer
All humans have the same set of ~25,000 genes
A mutation in any gene could potentially
result in a loss (or gain) of function resulting in
disease.
A “disease gene” is a “normal” gene which,
when mutated, results in disease.
The number of new disease genes identified from human genome data is disappointingly small.

14. Searching for “Disease Genes”: New Strategies
1) Look for multiple common mutations among members of populations in which a disease is prevalent.
2) Look for mis-expression of normal genes:
Rather than looking for mutations in genes, look for aberrant expression of genes.

15. Searching for “Disease Genes”: New Strategies
MICROARRAY
ANALYSIS
Used to compare gene expression in two different tissues. For example:
Expression levels in malignant tissue can be compared to expression levels in normal tissue for the same set of genes.

16. Each sequence corresponds to a specific gene.
MICROARRAY
ANALYSIS
(continued)
Short nucleotide sequences are synthesized directly onto a glass “chip”.
Each sequence corresponds to a specific gene.
Thousands of sequences can be spotted onto a single chip.

17. Isolate RNA from malignant lung tissue
Reverse transcribe to cDNA (red fluorescent label incorporated)
Isolate RNA from normal lung tissue
Reverse transcribe to cDNA (green fluorescent label incorporated)
Apply cDNA mixture to chip
Complementary base pairing between cDNAs and chip spots.

18. How is gene expression controlled on a global scale?

19. Darkly stained Regions = Heterochromatin
Chromatin Structure
Darkly stained Regions = Heterochromatin
Lightly stained Regions = Euchromatin
Drosophila Polytene Chromosomes
Whether or not a gene is expressed depends, in part, on whether it resides in heterochromatic or euchromatic region.

20. “Position Effect” w+ encodes red eyes in Drosophila
the gene is expressed
when it resides in a euchromatic region of the chromosome
When the gene is experimentally placed near a heterochromatic region, the heterochromatin spreads, silencing the gene.

21. Chromatin staining properties are related to nucleosome structure.
A nucleosome is made up of four different histone proteins, two copies of each.

22. Chemical tags on histone tails comprise a “Histone Code”
HAT= Histone Acetyl Transferase
Tags include:
methyl groups
acetyl groups
and others
HDAC= Histone DeAcetylase
Tags are either repressive or permissive with respect to gene expression.
Depends on type and location of tag.

23. In stem cells, many promotors exist in a “bivalent” state.
permissive mark
repressive mark

24. DNA methylation plays a huge role in regulation of gene expression
Promotor methylation usually = gene silencing
Methylation is established at certain CpG sites and maintained through subsequent cell divisions.

25. This regulatory system constitutes the Epigenome
Regulation of Histone Modification and DNA Methylation are inter-related

26. “Happy Puppet Children”
Angelman’s Syndrome
“Happy Puppet Children”

27. This region of chromosome 15 is paternally imprinted (silenced).
Angelman Syndrome caused by a deletion in chromosome
only if the deletion is inherited from the mother Explanation?
Marcus Pembrey:
Clinical geneticist at the Institute of Child Health in London
This region of chromosome 15 is paternally imprinted (silenced).
Epigenetic marks are erased/ re-established during gametogenesis.

28. Suggests environmental influence
Agouti gene methylated:
Mouse is normal.
Agouti gene unmethylated:
Mouse is obese
Prone to Cancer
Prone to Diabetes
Phenotype can be reversed by a methyl- supplemented diet.
Genetically identical mice develop different phenotypes depending on methylation status of the Agouti gene.
Suggests environmental influence
on epigenome.

29. Carlos Aizenman
Brown University
Department of Neuroscience

30. Rat pups that experience maternal care have reduced methylation of the Glucocorticoid receptor gene promotor.
The pups acquire the behavioral trait without the trait being passed through the germ line.

31. Reduced expression of GR gene in
suicide victims who had experienced abuse as children
Increased methylation of GR gene promotor in
suicide victims who experienced abuse as children.

32. There is some evidence that some epigenetic marks survive gametogenesis.
This suggests that some acquired traits could be inherited.
Jean Baptiste Lamarck

33. Maternal Care Smoking Diet
Can our behaviors influence the quality of the genome we pass on to our children?
Maternal Care
Smoking
Diet

34. Please pick up your exam in Biomed B25
Details of how letter grades will be determined are on web site.
Jody’s Office Hours
3:00 – 5:00 PM today.
Please pick up your exam in Biomed B25

36. Instructions for Regrade Requests are on Exam Web Page — drop off written request outside SFH Requests must be made by 4/3/13, 14 days from today.

37. Bisulphite Sequencing
Prior to sequencing, a DNA sample is treated with bisulphite.
Result:
Unmethylated cytosine residues are converted (by deamination) to Uracil.
Methylated cytosine residues are not deaminated.
We know this C was previously unmethylated because it was converted to a U upon bisulphite treatment.