Genetics (IASM1-7) Flashcards
The central dogma?
DNA > RNA by transcription
RNA to protein by translation
Define transcription (state its location)
The 3 stages of transcription?
What types of post-transcriptional mods are there for the mRNA strand produced?
Where does the mRNA synthesized during transcription go
synthesis of mRNA copied from DNA base sequences by RNA polymerase II in the nucleus
stages: initiation, elongation, termination
mods: 5’ capping, adding 3’ poly-A tail, splicing to remove introns
mRNA transcript moves out of nucleus into cytoplasm for translation
Process of initiation in transcription?
RNA polymerase II binds to promoter region (at the TATA box)
Protein transcription factors bind to promoter region
RNA polymerase unwinds DNA double helix and initiates transcription
Function of the promoter region, relative location to the gene to be transcribed and functions of transcription factors?
Promoter region:
contains promoter sequences (TATA box) or RNA polymerase binding, also serves as binding site for transcription factors
location: 25-35 base pairs upstream of gene to be transcribed
transcription factors: proteins that can control rate of transcription by promoting or repressing transcription
Process of elongation?
mRNA synthesized from 5’ to 3’ direction
RNA polymerase 2 moves along the antisense (template strand), adding new mRNA nucleotides to the 3’ end of the strand
free mRNA nucleotides (nucleotide TRIPHOSPHATES) aligned opposite to their exposed complementary DNA partner are joined together covalently by using the energy from cleavage of additional phosphate groups
Coding strand goes around the “outside and not actually directly involved
Process of termination?
RNA polymerase II reaches the terminator region on DNA (RNA DOES NOT have the specific sequence to direct RNA polymerase II to terminate at a specific location so the terminator region is DNA)
enzyme and nascent mRNA strand detach from DNA template, DNA rewinds
Then post-transcriptional modification: 5’ capping, adding 3’ polyA tail, splicing to remove introns
How are the post-transcriptional mods done
5’ capping: 7-methylguanosine attached to the 5’ end of the hnRNA to form 5’ cap
3’ polyA tail: polyA tail attached to 3’ end by Poly A polymerase
intron removal: using proteins to form spliceosome for introns to be spliced (only exons remain)
Purposes for post-transcriptional mods?
Protect mRNA against early degradation (increased stability)
facilitate translation
Process of translation?
- RNA passes out from nucleus to ribosomes in cytoplasm where translation takes place
- The ribosome has a large and small subunit which assemble around the mRNA
- Transfer RNA (tRNA) carries individual amino acid
- Codon on mRNA matches anticodon on tRNA
- A site, P site then E site as sequence that tRNA binds at ribosome : APE
- Protein synthesis proceeds with finished amino acid chain emerging from ribosome then folding into conformation
The structure of hemoglobin and no.of genes coding
tetramer (2 alpha subunits, 2 beta subunits)
alpha protein subunits: coded by HBA1 and HBA2 genes
Beta subunit HBB1 coded by HBB gene
3 genes in total
Cause of phenotype in sickle cell anemia?
Hemoglobin gene mutation in HBB subunit (HBB gene mutated to HbS gene)
glutamic acid replaced by valine
valine is hydrophobic so tends to stick together and clump up to avoid aqueous environment
tends to form fibers
cell shape distorted
Why is the sickle-cell phenotype still prevalent in Sub-Saharan Africa
Risk allele helps protect against severe malaria
Cause of thalassemia phenotype and its inheritance pattern?
Produce no or too little hemoglobin (alpha or beta
thalassaemia)
Autosomal recessive
Inheritance pattern of cystic fibrosis?
The symptoms of cystic fibrosis?
Cause of cystic fibrosis phenotype?
Autosomal recessive
Symptoms: salty skin, poor growth, thick sticky mucus, chest
infections
Absence of phenylalanine in CFTR gene (codes for CFTR that
transports chloride across membranes)
Delta F508 (Phe)
Core histone proteins in the nucleosome?
H2A, H2B, H3, H4
Some numerical data about DNA:
1) width of the DNA helix?
2) length of major and minor groove for each turn of the double helix?
3) How many base pairs per turn
4) degrees per rotation of base
1) 20A
2) 34A, 3.4A
3)~10.4
4) 36
Cause of spinal muscular atrophy phenotype?
Exon 7 not included → cannot make SMN1 and so rely on SMN2 to make protein→ shorter SMN Protein ⇒ non functional
CRISPR-Cas9 mechanism in bacteria and its function?
Immune response preventing viral infection
Guide RNA
■ matching sequence with RNA complementary to the DNA of
the invading virus
Tracer RNA
■ constant region of RNA that scaffolds with the Cas 9 protein
Guide RNA + Tracer RNA: sgRNA (single guide RNA)
complex + enzyme (nuclease) ⇒ Cas9
○ RNA locks onto the PAM of viral DNA, Cas9 unzips DNA and matches it to target RNA
○ Cas9 cuts DNA, cell tries to repair the gene but the process is error prone
The applications for dCas9? (meaning of dCas9)
dCas9: dead Cas9 with only the binding activity but not endonuclease activity
1) CRISPR can be used to add transcription factors to promote transcription
2) or CRISPR can be used to directly bind Cas9 to a DNA sequence to silence that gene
3) or attach fluorescent proteins to observe DNA loci activity
Overview 1 clinical trial using CRISPR-Cas9
Curing Leber congenital amaurosis: inherited blindness caused by mutation of CEP290 gene coding for CEP290 protein that helps retinal cilia properly function
CRISPR-Cas9 can help correct the mutation by gene editing
Insulin action that causes glucose absorption? And the consequences?
● Insulin binds to receptor
● The glucose transporter is inserted into the membrane to allow glucose shuttling
● There are more glucose transporters embedded in the membrane
● [Downstream: glucose transporters are transported up into membrane]
● Change in conformation of intracellular domain of insulin receptor
● Increase uptake of glucose into liver cells/ muscle cells
● Increased glycogen synthesis (glycogenesis) as a response to increase in insulin
Amino acid groups?
Nonpolar aliphatic: GAVLIMP (glycine, alanine, valine, isoleucine, leucine, methionine, proline)
aromatic: WFY (WIFI) (Phenylalanine, tyrosine, tryptophan) (TYROSINE IS POLAR)
Polar uncharged: STC, NQ (slay the children, new quilts) (serine, threonine, cysteine, asparagine, glutamine)
Polar positive (basic): HRK (徐克 LAH）（histidine, arginine, lysine)
Polar negative (acidic): DE (Aspartic acid, glutamic acid)
Essential amino acids?
PVT TIM HiLL
Phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histamine, leucine, lysine
Which groups of amino acids form the protein centers and why
nonpolar aliphatic and aromatic as they are hydrophobic
why can cysteine be considered nonpolar
can methionine form disulphide bridge
Disulphide bridges can form between 2 cysteines BUT the polarity of the disulphide bridge is so weak it can be considered nonpolar/hydrophobic
methionine CANNOT form disulphide bridge
The mutation causing cystic fibrosis?
ΔF508 (loss of phenylalanine at amino acid position 508)
Which chiral form of amino acids do proteins in the body use
L-form
Define the levels of structure of proteins
primary structure: sequence of amino acids from N-terminal to C-terminal (including post-translational modifications eg phosphorylation)
secondary structure: local conformation of backbone due to interactions between atoms (h-bonds) in the backbone
tertiary structure: overall 3D structure of one polypeptide due to interactions between the side chains of amino acids
quaternary structure: overall final 3D structure of a protein made by multiple polypeptides
Numerical data for alpha-helix:
1) how many amino acids per turn?
2) length of helix per turn
1) 3.6 amino acids per turn
2) 5.4A per 360-degree turn
The 2 forms of beta sheet? Describe in one word their structure
parallel and antiparallel, pleated
The types of proteins in body?
Fibrous, globular
Features of fibrous proteins?
Long, extended and repetitive sequences
Structure of collagen? Its amino acid components?
Right-handed triple helix, made from proline, hydroxyproline, glycine
Disease name of type 1 collagen mutations and its consequences?
Osteogenesis imperfecta, increased bone fractures and collagen defects
Explain the need for Vitamin C in making collagen
Vitamin C needed to keep Fe2+ reduced to maintain prolyl-4-hydroxylase activity to produce hydroxyproline
Keratin structure?
2 alpha helices wrapping around each other - coiled coil structure
How does hair curling work
The tougher the keratin the more cysteines involved in disulphide bond so to curl hair first reduce cysteine to break disulpide bridges and then curl, and then reoxidize some cysteines
Globular proteins as oxygen carriers? (2 of them)
Hemoglobin, myoglobin
What is at the center of heme group and its function
Fe2+ at middle of heme to bind with oxygen
Myoglobin vs Hemoglobin - compare their structures and how many oxygens can bind
myoglobin: monomeric with single heme molecule, only 1 oxygen binds
hemoglobin: tetrameric, 4 heme molecules, 4 oxygen can bind
How is positive cooperativity in hemoglobin driven by oxygen
1st oxygen binds to hemoglobin with low affinity
then the 2nd, 3rd and last oxygens bind with increasing affinity
because hemoglobin changes structurally from T (tense) state to R (relaxed) state which increases its affinity for oxygen
Importance of positive cooperativity?
Enables saturation of hemoglobin with oxygen in lungs but allows oxygen to be released to tissues as pO2 is very high in lungs and low in tissues (think about to pO2 graph)
What does carbon monoxide do to oxygen (in terms of oxygen absorption)
at what percentage of COHb can cause coma
why can some anemic patients still survive even with lower lung and tissue pO2
CO disrupts hemoglobin cooperativity and locks it into high affinity R state
Hemoglobin cannot release oxygen to the tissues - 50% COHb causes coma
some anemic patients can still survive because hemoglobin can still pass oxygen to myoglobin in tissues (positive cooperation still intact)
How many base pairs in the human genome
3 billion
In terms of ploidy what is the human genome
diploid
Process of inheritance?
Gametes (sperm, oocyte): haploid > fertilization, sperm + oocyte > diploid > somatic cells: diploid
Properties of functional genome?
Organized, usable for regulation and expression, stable, must be copied accurately to next generation of cells
What principle is DNA sequencing based on
○ Addition of complementary bases to template strand (elongate chain)
○ Complementary bases added onto the 3’OH group of the previous base
The types of DNA sequencing (the 2 big types)
Classical Sanger sequencing, Next-generation sequencing
How can next-generation sequencing be divided
what types of each form of NG sequencing are there
Short reads and long reads
sequencing by synthesis (Solexa/Illumina)
Nanoball sequencing (Complete Genomics/BGI)
pH sensing (Ion Torrent/ThermoFisher)
Long reads: single molecule real time sequencing (by dye labels or nanopore sensing)
Steps for sequencing by synthesis?
- DNA sample prep - dna fragments with adaptors, barcodes
- amplification - clusters of sequences (WITH PCR)
- sequencing - labeled nucleotides, imaging (when fluorophores attached and then the base identified they are cleaved and removed, regenerating a 3’OH for the sequencing cycle to continue)
A single flow cell can produce billions of ~150 bpDNA sequences
Steps of detecting genetic variation?
Detecting genetic variation
○ Sequence data → align to reference genome → call sequence variants
Types of genetic variation and their meanings? Arrange them according to how large the alteration is. Also give examples if possible
Copy number variation (largest effect)
Loss or gain of whole or arms of chromosome
Example: Down syndrome, trisomy 21
Structural variation: Large alterations
Defined as including:
1) Deletions
eg Facioscapulohumeral Muscular Dystrophy (FSHD) - deletions of large amounts of D4H4 gene copies (of 30-3000kb) (leaving only <11 copies)
2) Duplications
3) Inversions (reinserted in the opposite orientation)
4) translocations (sequence translocated from one chromosome to another; or different location on same chromosome)
eg FSHD
Sequence level variations (small alterations)
1)Single nucleotide polymorphisms (SNP) - nucleotide variation
eg Sickle cell anemia (GAG → GTG: produces HbS (glutamic acid to valine)
2) Indels - insertion/deletion mutation (frameshift mutation)
Significance of genetic variation?
Underlies most phenotypes
Affects large or small parts of the genome
Many disease associated variants directly alter gene sequence resulting in mutant proteins
Genetic variants can also affect gene expression
