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159 Cards in this Set

  • Front
  • Back
Huntingtons disease, frgile x syndrome and myotonic dystrophy are all examples of what type of genetic disease?
Trinucleotide repeat expansion diseases.

Caused by unstable mutations of triplet repeat where the repeat number correlates with the severity of the disease.

"Genetic anticipation" causes progressively earlier age of onset.
Which bases are purines or pyrimdines?
A and G purines
C, T and U are pyrimidines

A binds to T or U (2 H-bonds)
G binds to C (3 H-bonds)
What type of bond exists between the base and sugar of nucleic acids?
N glycosidic bond
What types of bonds bind nucleotides together?
Phosphodiester bonds
What is the difference betwen an endonuclease and exonuclease?
Endonucleases cleave internal phosphodiester bonds.
Exonucleases cleave the last phosphodiester bond on the 3' or 5' end.
What are the main conformations of double helical DNA?
B-DNA (Watson and Crick) is the most common.

A-DNA
Z-DNA
What trinucleotide repeats can cause triplet expansion diseases and how?
CTG, CGG: form stable hairpin structure

GAA: forms stable triplex structure
In the tertiary structure of DNA, what is the difference between a positive and negative supercoil?
Positive is a left handed superhelix.

Negative is a right handed superhelix.
What is a DNA topoisomerase?
An enzyme that can change DNA tertiary structure but amazingly DO NOT require ATP.

Topo 1: cuts a single strand.
Topo 2: cuts both strands.

DNA gyrase is a special type of bacterial Topo 2 which can add a negative supercoil into relaxed DNA. It is the target of fluoroquinolones.

Camptothecin and Etoposide, that respectively target eukaryotic topo I and topo II, are used in chemotherapy.
The complex of protein with nuclear DNA is called ...
Chromatin
What is the basic unit of chromatin?
A nucleosome.

A nucleosome contains ~146 bp of DNA, and two copies of each core histone (H2A, H2B, H3, and H4).
DNA is wrapped 1 3/4 turns around the outside surface of the histone octamer.
What is a polynucleosome?
“beads-on-a-string” structure of chromatin.

The region between the nucleosomes (the "string") is called the linker region and is associated with histone H1.
What is a chromatosome?
The bead.

a nucleosome + H1 +166 bp to complete a complete turn since the DNA in the nucleosome only covers 1 3/4 of a turn.
What is a chromatin fiber or solenoid?
The beads on a string braided together by the interaction between the H1 proteins associated with the chromatosomes.
How do chromatin fibers (solenoids) interact to form a chrmosome?
The solenoid coils upon itself, resulting in the formation of large DNA loops.

Large loops of coiled solenoid are anchored to scaffold proteins and radiate outwards from the center (radial loops) to form a chromosome.
In order, what are the phases of the cell cycle?
S,G2,M,G1
Why is DNA replication called semi conservative?
Each daughter molecule has a new strand and a strand from the parent molecule.
What is an origin of replication?
An AT rich region (less H bonds) where bidirectional replication begins.

Parental strand seperation is catalyzed by DNA helicase and creates two replication forks at the origin.

Single-strand DNA-binding (SSB) proteins bind to DNA strands at the origin to keep them apart and protect them from nucleases.
How do inhibitors of Herpes simplex virus (HSV) work?
They act on viral helicase and primase.
How does DNA synthesis begin?
An RNA primer is synthesized by the RNA polymerase, primase, since DNA polymerase cannot initiate synthesis on a single strand.

RNA primers provide a free 3'-OH group that accepts the first deoxyribonucleotide.
Which direction do newly synthesized strands made out of dNTP's by poylmerase run?
5'-3' direction
What do we mean when we say DNA replication is semi-discontinuous?
The leading strand is synthesized continuously toward the replication fork.

The lagging strand is synthesized discontinuously in short Okazaki fragments directed away from the fork.
Which prokaryotic DNA polymerases have 5' to 3' polymerase activity?
All of them. (POL I, POL II and POL III)
Thats why they are called DNA polymerases.
Which prokaryotic DNA polymerases have 3' to 5' exonuclease activity?
All of them.
(POL I, POL II and POL III)
Which prokaryotic DNA polymerases have 5' to 3' exonuclease activity?
Only DNA POL I.
How does chain elongation occur chemically?
DNA polymerase catalyzes the nucleophilic attack of the 3'-OH terminus of the growing DNA chain on the innermost phosphate group of an incoming dNTP.
The formation of a phosphodietster bond releases a pyrophosphate group.
HOW does AZT work?
It is a nucleotide analog that incorporates into newly forming HIV DNA and stops chain elongation.
What do we mean by proofreading?
The 3' to 5' exonuclease activity of DNA polymerases allows the removal of nucleotides that are not complementary to the template.

After DNA replication, repair mechanisms can replace mismatched bases that escaped proofreading.
What happens when DNA POL III which is happily elongating a chain encounters an RNA primer?
DNA Pol I gets involved.

5' to 3' exonuclease activity removes RNA primer from Okazaki fragments.

5' to 3' polymerase activity synthesizes DNA to fill the gap.

3' to 5' exonuclease activity allows it to proofread the new DNA chain.
What is the function of DNA ligase?
It joins Okazaki fragments
How is synthesis on the leading and lagging strand coordinated?
The polymerases working on both strands are assembled into a large complex called a replisome.
How is DNA replication initiated in eukaryotes?
Eukaryotic DNA is replicated from many origins (ORI's).
Which eukaryotic DNA polymerases have have 5’ to 3’ polymerase activity.
All of them.
Which of the eukaryotic DNA polymerases involved in DNA replication has exonuclease activity?
Pol δ (delta)
3' to 5'

Unlike prokaryotic polymerases, this thing can synthesize leading and lagging strands as well as gap synthesis. (very fancy)
In what major ways is eukaryotic DNA replication different from prokaryotic DNA replication?
Much larger amount of DNA.

More complex DNA polymerases.

DNA association with histones.

The presence of telomeres
How is the nucleosome handled in eukaryotic DNA synthesis?
Nucleosomes are displaced as the replication fork advances.
Histones remain loosely associated with the parental DNA.
New histones synthesized simultaneously with DNA replication.
Nucleosomes reform rapidly behind the replication fork.
What is a telomere?
Short G-rich repetitive DNA sequences.

In humans, (TTAGGG)n at ~1000 copies/telomere

Prevent the loss of terminal sequences during replication.

Protect chromosome ends from fusion, and from being recognized as broken DNA and degraded.
What is telomerase?
Telomerase is a ribonucleoprotein complex (RNA + protein) with reverse transcriptase activity (hTERT): it synthesizes DNA using its RNA subunit as the template.

It adds short DNA repeats to the 3'-ends of linear chromosomes. This extension allows room for a primer to bind and initiate lagging-strand synthesis on the other strand, maintaining an appropriate length of chromosome ends and preventing the loss of coding sequences.

Telomerase has been Implicated in cell aging and cancer.
Most somatic cells do not express telomerase but
in cancers, telomerase is re-activated, allowing indefinite cell division and cancer progression.

Telomerase inhibitors can limit cancer cell proliferation.

Dyskeratosis congenita is caused by reduced telomerase activity and effects highly proliferative tissues like hair follicles,gut, skin, bone marrow.
What is xeroderma pigmentosum?
Relatively rare autosomal recessive disease caused by a defect in DNA nucleotide excision repair. Caused by mutations in any of 7 XP genes.

Sunlight sensitivity as well as cigarette smoke carcinogen sensitivity.

High incidence of skin cancer.

Neurological abnormalities.
What sorts of mutations can happen in DNA?
Nonsense - change creates a premature STOP codon or eliminates a STOP codon.

Missense - introduces a different amino acid.

Frameshift - changes all subsequent codons.

Alteration of gene regulation - change affects control of gene expression.
What is a replication error?
A replication error creates a mismatched pair that may be corrected by replacing one base.
If uncorrected the error becomes a point mutation because it is fixed in one of the daughter duplexes.
One of the mechanisms for generating errors in DNA is spontaneous deamination. How does this work?
One base is changed into another by a deaminase.

If C is deaminated it becomes U. This happens about 100 times/cell/day.

If A is deaminated, it becomes hypoxanthine.

If G is deaminated it becomes xanthine.

T does not have an amino group to be removed.
One of the mechanisms for generating errors in DNA is spontaneous depurination. How does this work?
Removes a base from DNA
2000~10,000 purine loss/cell/day

Creates an “abasic” site
blocking replication and transcription which results in a deletion in the daughter strand.
Alkylating agents like methyl donors can cause mutations in DNA. How do they do this?
Methylation of a base causes it to change its properties and distorts the double helix. This allows incorporation of a mismatched (non-watson and crick) base pair leading to a mutation in the next generation.
How does radiation damage cause mutations?
UV radiation for example, covalently links adjacent pyrimidines along one strand of the DNA.
The dimer blocks replication and transcription.
What are the pathways for DNA repair?
Mismatch repair
Base excision repair
Nucleotide excision repair
How does the mismatch repair pathway work?
Recognition of mismatches relies on distortion of the double helix.

In E.Coli, the protein MutS recognizes the “incorrect” pair by detecting the structural distortion in the DNA.

The enzyme knows which one the correct base is in the mismatch because of the presence of methyl groups at GATC sites of the parental strand. The new mutant strand will not have had a chance to acquire these methylations yet.
In other words, the enzyme knows which strand is the original one based on GATC site methylation which could potentially be hundreds of base pairs away from the mismatch site.

The mut protein complex then cleaves the mismatched base with the aid of helicase. DNA ligase seals the nick to create the correct base pair.

In humans, the mechanism for recognizing the new strand is unclear because there is no GATC methylation.
The protein complexes in humans are called MSH proteins, MLS proteins and PMS proteins.
What is Hereditary Nonpolyposis Colorectal Cancer (HNPCC)?
It is a defect in mismatch repair

Mutations in Mut protein homologs are found in the majority of HNPPC patients.

Mutation rate sin these patients are elevated by 100- to 1000 fold.

Accumulation of mutations eventually leads to tumor formation.
What is the base excision repair pathway?
It is an essential process that repairs single base damages including deaminated bases, depurinated bases (Abasic sites) and methylated bases.
What are the steps in the base excision repair pathway?
Glycosylase cuts the N-glycosidic bond between the sugar and the base. Removal of the base creates an AP site

AP endonuclease cuts the 5’-phosphodiester bond at the AP site.

AP lyase removes the sugar by cutting the 3’- phosphodiester bond.

DNA polymerase fills the single nucleotide gap

DNA ligase seals the nick
What is the nucleotide excision repair (NER) pathway?
Removes a wide variety of lesions, photoproducts and chemical adducts especially large adducts or distortion of double helical DNA structure such as pyrimidine dimers.
How does nucleotide excision repair (NER) work?
Lesions recognized by XP proteins (Uvr proteins in E. coli). This is the defect in xeroderma pigmentosum.

Exconucleases make incisions 5’ and 3’ to the lesion.

Helicase releases the damaged oligomer (12-13 nt in E. coli; 27-29 nt in humans).

DNA polymerase fills gap.

DNA ligase seals nick.
What is homologous recombination?
Can occur anywhere between DNAs with high sequence homology but not necessarily identical.
Crossing over during human meiosis is a great example of this.

The process is catalyzed by recombinase proteins.
What is site specific recombination?
Joins two DNA segments with short but specific DNA sequences present on both participating DNAs and recombination occurs only at the specific sites.

Catalyzed by recombinases.

A good example is antibody diversity generation by V(D)J rearrangement mediated by RAG recombinases.
What is transposition?
Transposons are DNA segments that can move from one DNA molecule to another, or to a different location within the same DNA molecule (cut and paste).
The process is catalyzed by transposases which are encoded by the transposons themselves. This makes the transposon a self contained mobile genetic element.

The joining of two DNA segments requires specific DNA sequences at the ends of the transposing DNA, but does not require a specific DNA sequence in the recipient DNA.
Antibiotic resistance in bacteria often results from transposition of genes from resistant bacterial plasmids to normal bugs.
What is the structural importance of RNA being single stranded?
RNA can form secondary structures that are important for specific functions (e.g., transfer RNA clover leaf with an anticodon, T-loop and D-loop)
In broad terms, what is transcription?
Transfer of genetic data from the DNA (genome/chromosome) to mRNA.

Catalyzed by RNA polymerases as well as other factors that assist in RNA pol binding.

Occurs in the nucleus of eukaryotes

Requires an opening of the DNA sequence, so that only one strand (antisense) acts as the template.

Contains three processes
Initiation, elongation, and termination
How does a change in the level of gene transcription ultimately effect the amount of translated protein?
There are multiple other factors invloved but generally more mRNA means more protein.
How is transcription intitated in prokaryotes?
Recognition and initial bonding of a multisubunit RNA pol complex that has picked up sigma factor (which makes it a holoenzyme) to promoter DNA sequences which are usually just upstream (5’) of the transcription start site.

The holoenzyme seperates the strands at the AT rich pribnow box and transcription is initiated.

Note: RNA synthesis does NOT require a primer.

Stable DNA- RNA polymerase complex transcribes at about 40 nucleotides/second until a termination signal is encountered but this stage is generally called elongation.

In prokaryotes, transcription can occur simultaneously with translation (protein synthesis).
What are the characteristics of prokaryote promoter regions?
Promoters have a conserved consensus sequence; the most conserved region is called the core. Usually there is a -35 (35 bases from the gene) conserved region and also a -10 conserved region called the Pribnow box.

There is also a spacer region that is conserved in size (~17 bases in prokaryotes), but not content.

These regions are targets for RNA pol binding
How does termination of RNA transcription occur in prokaryotes?
Rho dependant termination:

Factor p (rho) binds to the growing mRNA strand and moves towards the oncoming RNA pol from behind it.
Rho-termination sequence at the end of the gene on the DNA causes RNA pol to pause and factor rho can catch up.
Factor p causes dissociation of the RNA pol and release of the mRNA.

Rho independant termination:
(more common than rho dependant).
Based on the secondary structure of the RNA, not the DNA.
Terminator sequence is a G C rich palindrome (same forwards and backwards) prior to 6-7 U’s.
The sequence causes the RNA to form a hairpin loop structure and dissociation ensues.
How does rifampicin, for the treatment of TB work?
works by binding to RNA pol and preventing transcription.
Specific to bacterial RNA pol, so it doesn’t stop transcription of the host.
How is transcription different in eukaryotes from prokaryotes?
Similar to prokaryotes, but much more complex.

DNA around genes is in a “looser” conformation to allow access of key enzymes/proteins.

Transcription factors must bind to promoter sequence prior to RNA polymerase (“Recruitment”).

Other sequences (enhancers) bind to activators to stimulate transcription.

Enhancers may be thousands of bp away from the promoter

Three different RNA polymerases exist in eukaryotes, each responsible for transcribing specific types of RNAs.
What are the three different RNA ploymerases in eukaryotes?
RNA POL I:
mostly transcribes ribosomal RNA.

RNA POL II:
The big one,transcribes mostly protein coding genes

RNA POL III:
Transcribes part of the ribosome, tRNAs and various small RNAs.
What is the promoter element in most eukaryotic cells?
the TATA box (similar to the pribnow box)
Other than RNA POL II, what is needed in the eukaryotic holoenzyme transcription complex?
General transcription factors, mediators, chomatin remodelling complexes and histone acetylases.
What is the "death cap" mushroom?
It produces a Toxin, a-amanitin which inhibits eukaryotic RNA pol II.
Symptoms start as mild GI symptoms, and can be followed by liver failure & death
What do we know about RNA polymerase 1 in eukaryotes and how it contributes to ribosome structure?
RNA polymerase 1 is specific for transcribing ribosomal RNAs (rRNA).

rRNA genes are located in nucleolar organizing regions (NOR), which are on the acrocentric chromosomes (13,14,15, 21 and 22 that have the little nub on them ).

Each transcriptional unit encodes three of the four rRNA subunits (28S, 5.8S, and 18S)

Hundreds of copies can occur in tandem in each NOR.

Post-transcriptional processing removes external spacers first, then internal spacers to degenerate 18S, 5.8S, and 28S rRNA
What does eukaryotic RNA pol III do?
Transcribes tRNA, 5S ribosomal RNA, and other small RNAs

Transcription factors for RNA pol III are usually located in the gene sequence, but can also be located 5’
How are transcribed tRNA's modified?
Several reactions must occur, but not necessarily in the same order every time

Introns must be removed

5’ portion must be removed by RNAse P (an endonuclease)

3’ portion must be trimmed by an exonuclease.

CCA must be added to the 3’ end by tRNA nucleotidyltransferase

Finally, the correct amino acid must be added to the CCA site.
What is the mechanism of RNA transcript elongation in prokaryotes an eukaryotes?
Even though prokaryotes and eukaryotes have different polymerases, the overall shapes of the catalytic regions are very similar.

Both polymerases “bend” DNA ~90° as it is transcribed

Both thread the DNA through channels in the enzyme

Both open a transcription bubble, where the two DNA strands are separated and the transcribed strand is exposed

Both form a temporary DNA/RNA hybrid

Both recruit nucleotides through a pore in the enzyme

Both provide an exit pore for the growing RNA strand

Both allow for reannealing of the DNA
Can eukaryotic RNAs be translated cotranscriptionally?
No.They must be stabilized and exported to the nucleus where translation will occur.

All RNAs must be processed before they are completely functional and the type of processing depends on the end function but in prokaryotes this can happen while the RNA is still being transcribed.
What is a CTD?
C-terminal domain.

Just after RNA transcription initiation (~25 bases) the C-terminal domain (CTD) is phosphorylated.

It recruits the enzymes responsible for mRNA 5’ capping, splicing, and poly(A) addition.
How is mRNA capping preformed?
Three enzymes modify the 5’ end by removing a phosphate group, adding a GMP molecule and finally
methylating the GMP.
How does mRNA splicing work?
The non coding introns are removed by small nuclear ribonucleoproteins (snRNPs).

The 5' end of the intron is cut first and annealed to an A in the RNA which is close to the 3' end of the intron. The entire thing is then clipped to release a characteristic lasoo or lariat shape piece of RNA.
What is heterogenous nuclear RNA (hnRNA)?
Intermediate forms of processed RNA.
Do prokaryotes preform RNA splicing?
No, it is unique to eukaryotes.

It allows for mutations to occur with less chance of a detrimental effect (the majority of most genes is intronic)

It also allows for more complex proteins to be created.
Which direction do RNA polymerases move in ?
RNA is always synthesized 5' to 3' which means the polymerase must move from the 3' end of the DNA strand to the 5' end.
What are the features of a mature mRNA transcript?
Poly A tail
Properly spliced mRNA
5' methylguanosine cap
What are the molecule sinvolved in the export of mRNA to the cytoplasm?
Heterogeneous ribonuclear proteins are bound to the RNA in the nucleus when it is mature.

One of these is the nuclear export receptor which bind snear the poly A tail and another is the CBC protein which binds to the 5' cap.

The complex is transported through the nuclear pore by an ACTIVE process.

In the cytosol, CBC is displaced by initiation factors for translation.

The nuclear export receptor is also displaced and recycled to the nucleus from the cytosol.
How many possible codons are there?
There are 4 nucleotides in the mRNA sequence, so there are 4^3 (64) permutations of the 3-letter code.
Which codons are stop codons?
UAA, UAG, and UGA
Which codon is a start codon?
AUG (Methionine)
How do we account for the fact that 61 tRNA’s are not required for 61 amino acids?
Most amino acids are carried by more than one tRNA species.

Some codons can be read by more than one tRNA (although they always carry the correct amino acid)

Accounted for by the “wobble” hypothesis: the third base pairing of the codon-anticodon is less strict than the first two.
What is a silent mutation?
One in which there is a DNA change, but no amino acid change.

The codon is different but still codes for the same amino acid.
eg. TAT and TAC both code for tyrosine so a change from T to C does not change the protein.
What is a substitution mutation?
A change in one or more bases that causes an amino acid change.
What is a nonsense mutation?
One which causes an amino acid codon to change to a STOP codon.
What is a frameshift mutation?
An insertion/deletion which shifts the reading frame and affects everything downstream of it.
What causes cystic fibrosis?
Mutations in the CFTR gene.


Mutations in the NBD1 domain of the CFTR protein are a subset of these.
How are amino acids bound their appropriate tRNA's?
Amino acids are “activated” by covalently linking them to the correct tRNA. The result is an adenylated amino acid bound to the tRNA molecule called an aminoacyl-tRNA.

ATP is consumed in the process.

The reaction is catalyzed by aminoacyl-tRNA synthetases which are specific for each tRNA-amino acid combination.

Proofreading occurs, and some aminoacyl synthetases can “deacylate” incorrect bonds before the reaction is completed to ensure fidelity.

The average rate of misacylation is 1/10,000 – 1/100,000 and is higher amongst amino acids with similar side chains like leu and val.
How are ribosomes adpated for protein translation?
Ribosomes consist of two subunits:
50S and 30S in prokaryotes
(from 70S parent molecule)

60S and 40S in eukaryotes
(from 80S parent molecule)

They are made up of ribonucleoproteins (RNA and proteins)

They are highly conserved throughout evolution as evidenced by the fact that subunit composition is similar in eukaryotes and prokaryotes

They can be found as free ribosomes in the cytoplasm which translate proteins for the cytosol, nucleus, or organelles.
OR
As membrane-bound ribosomes on the ER which translate proteins that will be secreted from the cell or remain in the ER/Golgi.
What is a polysome structure?
An assembly line of ribosomes that line up to work cooperatively.
How does translating mRNA into proteins work?
mRNA is used as the template and read from 5’ to 3’ by the ribosomes

Protein is synthesized from the amino-terminal to carboxy-terminal.

Same steps involved as with transcription:
Initiation, elongation, and termination.
What are the three parts of the large ribosomal subunit?
E site:“Exit” site
P site: Donor or “Peptidyl” site
A site: Acceptor or “aminoacyl” site
How is translation initiated?
eIF = eukaryotic initiation factor.

eIF 2 binds to the tRNA and eIF 4e/4g to the 5' end of the mRNA displacing the CBP cap.

Movement along the mRNA in search of the first AUG is facilitated by other initiation factors, which are powered by ATP hydrolysis.

When the AUG is found, eIF 2 and other initiation factors dissociate and the first aminoacyl tRNA binds.

The first peptide bond forms between methionine and the amino acid.

Elongation begins.
What is translation elongation?
Stepwise addition of amino acids to the growing peptide chain.
Uses peptidyl transferase to covalently bond adjacent amino acids.
How is translation terminated?
Release factor which mimics a tRNA binds to the A site of the ribosome and causes water to be transferred to the peptide chain, which releases it from the ribosome.

The ribosome dissociates, and is free to translate another mRNA.

The mRNA is also released and may be translated again.
Why do some cells differentiate into epithelial cells, hepatic cells, neurons, etc.?
Genes are regulated in different cell types to alter expression levels.
What is the most efficient point to control protein expression?
Transcriptional control.
How do transcription factors regulate gene expression?
Transcription factors regulate RNA pol II activation.
Where do transcription factors bind DNA?
On the outside of the double stranded DNA.
They localize to the 3D shape of specific sequences in the major groove because this is the only place where sequences actually impact structure.
Transcription factors typicall home to very short consensus sequences with a highly conserved core.

eg. GATA1 (TGATAG) and Oct-1-Pou domain (
How do transcription factors actually bind DNA?
Involves hydrogen bonds, ionic bonds, hydrophobic interactions, etc.

Certain DNA-binding motifs from TFs have been identified:

Helix turn helix motif:
Composed of two a-helices connected by a turn. The so called recognition helix is the part that actually binds the major groove and is on the carboxylic acid end of the molecule.
Some helix-turn-helix TFs have two motifs that bind to adjacent major grooves.

Zinc finger motif:
Characterized by two b-sheets, a turn, and one a-helix. Also contains a single zinc atom and is named for the aa that hold the zinc. CYS-CYS-HIS-HIS is a common one.
The alpha helix is the part of the zinc finger that actually binds the major groove.

Other motifs include b sheets, leucine zipper, helix-loop-helix, etc.
Transcription factors account for only part of the required molecules for gene regulation in eukaryotes. What else plays a major role?
Enhancer sequences and their activators exert significant control over expression levels.
How do activators regulate transcription?
Activators can alter the chromatin structure around the promoter by histone and nucleosome modifications.
They can make the promoter region more accessible to the necessary transcription factors and RNA polymerase.

Activators can exhibit transcriptional synergy which means the effect of multiple activators is much more than just additive.

Gene expression regulation is very complex though and in some instances transcription factors, activators and repressors can compete for the same sites which means concentration becomes very important.
To add to this, one gene regulatory protein can affect multiple genes simultaneously and/or respond to various endocrine signals.
What is DNA methylation?
A mechanism of gene regulation.
Promotor sequences which are G-C rich have CpG islands (1-2kb stretches with more than 50% C's and G's).
CpG islands typically indicate the promotor region of a gene.
The CpG islands are the targets for cytosine methlation which can affect the function of the promoter.
What is microRNA (miRNA)?
They are yet another mechanism of gene regulation.

Fairly recently discovered, but important for regulation of ~1/3 of all human genes

Typically 20-30 nucleotides in length.

Regulate mRNA sequences by targeting the 3’ end and “slicing” the transcript, making it susceptible to degradation
Argonaute and RISC proteins are involved in the process.
What percentage of the human genome actually codes for protein?
5-7%
What do we mean when we say that siblings are concordant?
They both have the trait.
What do we mean when we say that siblings are discordant?
Some have the trait and some dont.
When choosing something to study for genetic component what aspect of the phenotype should we keep in mind?
The more objective the phenotype, the easier it is to find the gene(s) that controls it
What is a candidate gene study?
Based on genes that are believed to be involved because of the biological function.

Independent of gene’s chromosomal location

Can also involve analysis of a gene pathway
What is a positional cloning study?
Identifying a disease gene based on its position in the genome, independent of its function.

Combines linkage analysis with physical DNA positions

Can identify a previously unknown gene or regulatory sequence.
What are the different types of polymorphisms that are useful in complex disease studies?
Microsatellites (simple sequence repeats, SSRs):
Usually 10-20 copies per repeat unit of 2-6 nucleotides (di-, tri-, tetra-, etc.)
Detectable by PCR.
Microsattelites are used mostly in genetic mapping.

Single nucleotide Polymorphisms (SNPs):
Single base changes
How do we genotype with microsatellite markers?
We compare two sequences that we know a specific location for and look for a length difference that will identify a different allele.
What is a Single Nucleotide Polymorphisms (SNPs)?
Naturally occurring genetic variation.

Single base changes (C to T, A to G, etc.)

Variants may have different frequencies in different ethnic populations.

May be responsible for slight changes in gene function or expression levels.
What is linkage?
The tendency of 2 or more loci to segregate together in a family.
The extent of linkage is a function of the physical distance between the loci and can be used to help map the genome.
What is the HapMap project?
An international effort to catalog genetic variation in multiple ethnic groups. Provides a reference for correlation between SNP's.

Can be used to select SNP's for genetic association studies.

Correlated SNPs are said to be in ”linkage disequilibrium” (LD). This actually means that they are highly correlated with each other despite the name.
What does it mean to “find a gene” for a complex disease?
Perform an association study and evaluate genotype frequency differences between cases & controls.

If the gene is far more common in cases than controls we assume we have "found a gene".

This says nothing about causation or mechanism though. It is merely correlation.
What is GWAS?
Genome wide association study.
Responsible for many of the new discoveries in complex diseases

Families not needed, since not looking for “linkage” (co-segregation of alleles and phenotype)

Summary data consists of p-values for selected phenotypes, allele frequencies, etc.

All results are added into dbGaP database.

Recently, privacy issues have caused this data to be pulled offline.
What is ORMDL3?
An asthma susceptibility gene.
What is copy number variation?
Certain regions of the genome vary in the number of copies of specific DNA sequences.

Can range in size from 10kb to 5Mb.

May be involved in disease susceptibility.
What is epigenetics?
The study of DNA changes that do not alter the actual DNA sequence.
eg. DNA methylation

Can be influenced by environmental exposures (chemicals, diet, etc.)

Changes can be transmitted from parent to offspring which we can't fully explain yet. Basically this means you can be obese because of something your grandmother ate.
How do small sample sizes effect genome association studies?
Yield more positive associations.
What is a chromatid?
Each chromatid is a double stranded DNA helix.

2 chromatids per chromosome – each identical
A human karyotype consists of...
22 pairs of autosomes
1 pair of sex chromosomes
What is an acrocentric chromosome?
13,14, 15, 21 and 22

Has sattelite stalks.
What are the arms of a chromosome called?
P-arms (short arms)
Q-arms (long arms)
What is euploidy?
Addition or loss of complete sets of chromosomes. eg. triploidy and tetraploidy
What is aneuploidy?
Gain or loss of a single chromosome usually because of a failure in meioses. eg. monosomies and trisomies.
What do we mean when we say a chromosomal translocation is balanced?
No loss or gain of genetic information but there is a position change.
Generally , there are no phenotype consequences (except when there is a position effect which causes gene disruption)

There are two types of translocations, robertsonian (13,14,15, 21,22) and reciprocal translocation.
What do we mean when we say unbalanced translocation?
Loss or gain or chromosome material is associated with unblanced translocations.

Abnormal phenotypes are likely.
Why is the modal number of chromosomes often reduced with a balanced Robertsonian translocation?
Fusion of two acrocentric chromosomes occurs.
Wolf-Hirshorn syndrome or greek warrior helmet syndrome is...
a 4p deletion
Cri du Chat syndrome is a deletion on ....
5p
Prader Willi syndrome (uncontrolled eating) is...
A deltion in paternal chromosome 15.

If the deletion is on maternal chromosome 15 the result is angelman ("happy puppet") syndrome.
The genetics of turner syndrome are...
45, X (formerly X0)
Kleinfelters phenotypes have a genotype....
47, XXY
What are some of the physical characteristics of fragile X syndrome?
Elongated face.
Large ears.
Autistic behaviour.

Often maternally carried.
Triplet repeat disease.
The risk of trisomy 21 between ages 35-39 is...
1:240

About the same risk as amniocentesis.
What happens in trisomy 16?
They typically do not survive to term.
An important point to keep in mind with reference to genetic statistics on neoplasia is that...
These are acquired abnormalities NOT constitutional.
CML or chronic myeloid leukemia is caused by...
The philadelphia tranlocation on chromosomes 9 and 22.
Lymphomas (like burkitt's) are typically caused by mutations in...
Immunoglobulin genes.
What is FISH?
Fluorescence In Situ Hybridization. Can be used to identify a wide variety of syndromes.

Each probe is specific to one region of a chromosome (pair), and is labeled with fluorescent molecules throughout it's length.

Step 1 - break apart (denature) the double strands of DNA in both the probe DNA and the chromosome DNA so they can bind to each other.

This is done by heating the DNA in a solution of formamide at a high temperature. 

Step 2 - the probe is placed on the slide and a glass coverslip is placed on top.

The coverslip is sealed with rubber cement.
The slide is placed in a 37 C incubator overnight to allow the probe to hybridize with the target chromosome.
        
What are subtelomeric FISH probes?
A powerful new tool
in detecting
cryptic telomeric
chromosomal rearrangements that often lead to mental retardation.
What is M-fish (SKY)?
Produces colored karyotypes that allow good visualization of translocations.
How is FISH useful in leukemia?
It can detect:

Translocations
Aneuploidy
Rearrangements
Amplifications
Etc.,
What is a breakapart probe for FISH?
A probe that binds to an entire gene and fluoresces if the gene breaks apart.
How is Synovial Sarcoma detected?
FISH
What is the Her-2 gene?
A predisposition gene to breast cancer.
How many chromosomes do most primates have ?
48
Chimps, Gorillas, Orangutans etc.
What are the steps in IVF?
Ovarian stimulation.

Transvaginal ultrasound guided aspiration.

Sperm immobilization.

Intracytoplasmic Sperm Injection (ICSI)
What is the trinucleotide repeat in fragile x syndrome?
CGG
What do the blot techniques place on a gel?
Northern=RNA
Southern=DNA
Western=Protein
What special nucleotides are used in Sanger sequencing?
2’3’-dideoxyribonucleotide (ddNTP)
How would we select a vector for cloning based on the size of the sequence we wan to clone?
Plasmids <10kb

Bacteriophage l vectors10~20kb

Cosmids 30~50kb

YACs (yeast artificial chromosomes) ~2000kb
How do we calculate the number of copies produced by PCR?
2^n

Where n is the number of cycles.
What is the trinucleotide repeat in huntington's disease?
CAG
How is human protein C, an anticoagulant, manufactured?
It is inserted for expression in pig milk glands and is isolated from the milk.