What is a single nucleotide polymorphism?
- A single nucleotide polymorphism is a single DNA base pair change present in at least 1% of the population, by itself a SNP usually has not clinical phenotype, most SNPs are sitting on noncoding regions
- Most SNPs are not sitting on the coding region
- 23andME and other companies use SNP microarrays
What is the normal sequence variation between humans?
- No two humans have an identical genome, any two humans vary in sequence by about 0.1-0.4% (3-12 million base pairs)
- Even monozygotic twins have slight variations that arise in cell division during early development
- Most variability occurs in the intergenic regions where most mutations are silent
What are mutations?
- Changes in DNA sequence that result in a phenotypical change- a trait or disease.
- Many mutations occur in protein coding regions, promoter, exons, or introns, UTRs and in ncRNAs; 50% of all disease causing mutations are in the non-coding regions of the genome
What percent of the genome is repetitive DNA?
53%, only 1.5% of the genome codes for mRNA
What two types of simple sequence repeats are there ?
mini-satellites and micro-satellites
mini-satellites are up to 25bp and telomers are the most common
micro-satellites are fewer than 13bp and the most common are CA repeats (no two humans have the same CA profile so CA repeats can be used to establish idenity in paternity and forensic cases)
What are LTR (long terminal repeat) transposons?
When you see LTRs in the genome, you know that sequence originated from a virus, it can be an exogenous retrovirus like HIV or it can be an endogenous retrovirus that you are just carrying around but has no disease phenotype
How are retrotransposons generated?
They are generated by reverse transcription of RNAs into cDNAS and insertion into the genome, RNA may be derived from a virus or form cellular RNA, insertion is usually random but may cause a mutation
What is the difference between LTR transposons and Non LTR transposons?
Non LTR are not viral
Where do non-viral retransposons come from?
These are not from a viral source but from your own RNA, your own RNA is modifying your genome
What are the two types of non-viral retrotransposons?
LINES and SINES
What are LINES?
Long interspersed nuclear elements, only Line 1 can transpose (has functional reverse transcriptase gene) and Line 2 and 3 are dead piece of DNA, hemophilia may be caused by Line 1 transposition
What are SINES?
short interspersed nuclear elements, borrow reverse transcriptase from Line 1, most common are "Alu" repeats (Alu repeats come from small non coding RNAs in the genome)
How do DNA transposons transpose in the genome?
DNA transposons are piece of DNA that can hop around the genome, the genome is not constant. DNA transposons transpose by replication and recombination.
- Conservative ( The transposon simply changes location in the genome with no increase in copy number)
- Replicative (stays in the same place, while a copy is inserted at a new genome position resulting in an increase copy number)
What is precision medicine?
- Precision medicine is a new approach to clinical practice that takes into account genetics, environmental exposure, age, and stage of development and health care system.
- Genomics can help with early detection, pharmacogenomics (monitor side effects, modify dosages) and genetic counseling.
What are complex genetic diseases?
Include most common diseases (breast cancer), no single variant is fully penetrant (multiple genese involved), environment/development/aging factors influence disease phenotype
What is the structural feature of a typical human gene?
pre mRNA is in the nucleus and mature RNA is in the cytoplasm [start and stop codons only function in the cytoplasm]
What is a promoter?
The area where RNA polymerase hops on
- gene expression is regulated the most at the promoter (if you don't need a lot of RNA, they don't have strong promoter but if you want a lof RNA then you have a strong promoter. Nature tweaks with these sequence and with regulatory componets in order to increase or decrease the activity of these promoters)
What are the two parts of a promoter?
Promoters have two parts - core promoter and regulatory promoter.
- The core promoter (TATA box, INR, DPE) are absolutely essential for RNA polymerase II to work but the regulatory promoter are sequences that are not absolutely necessary to begin transcription but enhance activity. It can happen that the core promoter is so weak that you could hardly detect a transcript from it but the regulatory parts bring int into biological important range.
How distinguish an RNA polymerase II promoter?
You look for a TATA box, but only 25-30% of RNAPII promoters have a TATA box
What does a eukarytoic RNA polymerase II recognize?
RNAPII doesn't recognize any DNA sequences, it's recognizing proteins bound to the promoter
What is the most important part of the promoter for RNAPII?
The most important component is the place where transcription starts, the initiator region, the other stuff could be there or could not but the initiator region is the only region in the promoter that has to be there because that’s where transcription actually starts.
What is the role of TFIID?
A set of proteins first find the promoter and these are called basal transcription factors (10 or 12) and they start to sit down on the promoter. The most important one is TFIID because it recognizes the earliest parts of the core promoter, it’s the one that makes first contact with the promoter. When it comes in, it starts bringing in the other transcription factors, they start assembly and come together with RNAPII and there’s this huge pile of proteins sitting there. Parts of the TFIID that see the promoter, they have different proteins that are looking at each one of those different elements. If you knock out TATA box, it’s still got contact with INR. If you knock out the downstream promoter element, you still have contact with TATA box and INR. Having these multiple contacts with the different components really solves your problem of how promoters can be so varied.
How does a far away (distal) promoter element regulate a core promoter?
This distance between the core promoter and some upstream activation sequence can be thousands of nucleotides (up to 40K). This happens by the magic of being able to loop DNA. You have the regulatory site making contact with core promoter via a special molecule called mediator.
How do snRPS work in splicing reaction?
snRNPs (pronounced "snurps"), or small nuclear ribonucleoproteins, are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs. The action of snRNPs is essential to the removal of introns from pre-mRNA, a critical aspect of post-transcriptional modification of RNA, occurring only in the nucleus of eukaryotic cells.
What does alpha-amantin do?
Alpha-amanitin si a powerful toxin (produced by mushrooms) and it's a strong inhibitor of RNAPII
How is translation initiated?
The ribosomes get on the 5' end and they scan to the AUG. You can regulate the scanning by having translation repressors that bind somewhere in the UTR or inhibit the ribosome form getting on in the first place and keep things from getting to AUG. (ex Iron Response binding element that can do this)
What are nonsense mutations?
A mutation that results in one of the three stop codons (UAG, UGA, UAA) because reading frame is shifted. A common misconception is that truncated proteins will accumulate due to nonsense mutations. Usually mRNA is degraded by nonsense mediated mRNA decay. You don't want to make a truncated protein becasue they compete with other proteins.
What are synonmous and nonsynonomous mutations?
Non synonomous mutations change the amino acid that is being coded for but synonmous do not
How do activators work (transcription)?
You can have activators that don’t touch anything but are involved in remodeling the chromatin that’s in front of the RNA polymerase so essentially saying “get out of the way”. The best way to think about this is that if you have a string of beads and you don’t have all the string covered with beads then you can slide those beads around; these chromatin remodeler proteins and associated proteins can move the chromatin away so RNAPII can access the promoter region You can have activators that don’t touch anything but are involved in remodeling the chromatin that’s in front of the RNA polymerase so essentially saying “get out of the way”. The best way to think about this is that if you have a string of beads and you don’t have all the string covered with beads then you can slide those beads around; these chromatin remodeler proteins and ssociated proteins can move the chromatin away so RNAPII can access the promoter region
How do transcription factors function as dimers?
Myc is an oncogene and if you make too much Myc then you have too much cell proliferation. Transcription factors don’t like to act alone they usually homodimers or heterodimers. Myc is partnered with its friend Max and when Myc and Max get together, they are an activator and they bind to a region upstream of the promoter, this friendship is okay and everything goes alright. However there is also another protein called Mad, and Mad is considered a repressor protein. Mad (not always expressed) binds to Max more tightly then Myc, then you have a MadMax dimer but when MadMax binds to the same sequence the gene is no longer activated.
How do epigenetics modify gene expression?
There is no change in DNA sequence. There is histone modification - changing the affinity of the histones to the DNA, you are changing the accessibility of the genome to regulation. There is also DNA methylation - C(phosphate)G regions are sites of methylation.
What does histone acetylation do?
It activates transcription.
What does histone deacetylation do?
It deactivates transcription (gene silencing).
What does DNA methylation do?
DNA methylation turns off genes by attracting histone modification enzymes and remodeling the proteins. Methylation attracts HDACs, compaction of the chromatin near the promoter.
What is alternative splicing?
Transcription regulations is the most important part of gene regulation, the second most important is alternative splicing. Splicing occurs when the splicesome identifies the 5’ prime splice junction and the 3’ prime splice junction of the intron and forms a lariet structure and and cleaves the 3’ splice site in order to release the intron as a lariet and fuses the two exons together. Alternative splicing occurs in a tissue specific manner. The same gene can code for two different proteins in two different tissues depending how it's spliced.
How are microRNA generated and regulated translation and mRNA stability?
Come from the nucleus, sometimes from introns. They bind to specific mRNAs the complementary sequences (3' UTR) and inhibit translation or cause cleavage.
- Fine tine expression of mRNAs in cell differentiation, development, growth and proliferation. They are critical in regulation of stem cells.
What does mismatch repair do?
repairs the mismatched nucleotide on the new strand
What does base exclusion repair do?
resects and replaces SMALL lessions (modified bases)
- Usually caused by metabolic damage this system repairs small lesions. The BER complex scans along, cuts out the bad, and re-synthesizes it
What does nucleotide excision repair and interstrand crosslink repair do?
repairs BIG helix distorting lesions
- repairs major helix distoritions usually caused by UV, or chemical agents. The complex chews up a large section of DNA (20-30 nts upstream of the problem) and re-synthesizes it. This complex is coupled with transcription.
How are double strand breaks and interstrand crosslinkds repair?
homologous recombination (HDR) and non-homologous end joining
Typically caused by ionizing radiation these breaks are repaired in two ways:
- (preferred way) homologous recombination: there is a recombination with the other chromosome to produce a nice restored full set. Ataxia Tel.
- End rejoining : this is where breaks will randomly join with other breaks. This can commonly cause translocations (example BCR-Abl). BRCA1/2 are important for homologous recombination complexes. And the DS breaks are recognized by poly(ADP)ribose which binds and acts as a signal to recruit repair pathways.
What causes Lynch Syndrome?
Caused by errors in Mismatch repair (MMR)
AD (aka hereditary nonpolyposis colon cancer HNPCC) 5% of all colon cancers, due to pathologic variations of mismatch repair genes : MSH2/6, MLH1, PMS2. Does not typically present with the extensive polyps of FAP (another colon cancer condition)
How do the DNA polymerases work?
Polymerase alpha Primase Complex Initiates DNA Replication and DNA Polymerases delta/epsilon Carry Out Elongation
What causes Xeroderma pigmentosum?
AR Caused by defect of nucleotide excision repair (NER), leading to massive risk of skin cancers, and other diseases. Any of the 8 genes responsible. Those afflicted avoid the sun at all costs, giving rise to the moniker “children of the night”.
What causes Ataxia telangiectasis?
AR result of pathologic mutations in the ATM protein of the homologous repair (HR) pathway. This causes ataxia (loss of control of muscles) in infancy, and telangiectasia (where blood vessels trace red, spider web like patterns visible on skin). Patients with this are immunodeficient, predisposed to other cancers, sensitive to other forms of ionizing radiation such as X-rays.
What causes Werner syndrome?
AR mutations in the WRN gene (helicase involved in HDR) patients with this disease are simultaneously short of stature, yet suffer from premature aging and all the consequences thereof.
What causes familial breat/ovarian cancer?
AR BRCA1/2 genes function in homologous repair. When mutated, have high association with breast, ovarian, and potentially prostate cancers as well.
What cause bloom syndrome?
AR mutations in the BLM gene (produces a WRN like helicase, also used in HR). Patients have similar physical and mental developmental delays. Phenotype also has a skinny face, sun sensitivity, butterfly shaped facial rash. Patients immunodeficient, at risk for cancers, esp. Leukemia.
What causes fanconi anemia?
DNA interstrand crosslink repair
Acute myeloid cancers. Defective recombinational DNA repair due to mutation in one or more of the following genes: FANCA, C,D2,E,F,G
Transcription vs Translation
During transcription, the entire gene is copied into a pre-mRNA, which includes exons and introns. Duringthe process of RNA splicing, introns are removed and exons joined to form a contiguous coding sequenc
How does DNA replication happen in humans?
Human gene replication is initiated at multiple origins of replication. An RNA polymerase called primase lays down a primer sequence for DNA pol to bind to and start synthesis. DNA synthesis complex needs a helicase to unzip DNA and a topoisomerase to unwind DNA. In addition DNA pol is locked onto the DNA by a sliding clamp protein. There are two types of synthesis: The leading/long strand and the lagging/short strand. DNA is always synthesized in a 5’ to 3’ direction. The lagging strand synthesis occurs in short segments called okazaki fragments which are then ligated together.
Why and how are telomeres added to the ends of chromosomes?
To solve the end of translation problem. With each replication event, the ends are shortened by a few bases. Telomerase insures that this is repetitive DNA and not useful DNA