Biochem Random Flashcards
Where does de novo purine/pyrimidine synthesis take place?
Cytosol
Purine de novo pathway
Ribose5P -> PRPP -> IMP -> either GMP or AMP
How do you make uracil?
Deamination of cytosine
Which nucleotides have a higher melting point and why?
C and G do bc they are connected via 3 hydrogen bonds while A and T are only connected by 2 H bonds
Which histones make up the core in the nucleosome?
H2A, H2B, H3, H4: two of each of these
What is the role of H1?
to link different nucleosomes together
Which AAs are needed to make purines? What additional substances are needed to make purines?
Glycine Aspartate Glutamine (GAG) are the AAs
You also need tetrahydrofolate and CO2
What is needed to make pyrimidines?
ASPARTATE, Glutamine, CO2, ATP
Pyrimidine de novo pathway
glutamine + CO2 + ATP -> carbamoyl phosphate THIS IS RATE LIMITING STEP -> orotic acid + PRPP -> UMP -> UDP -> CTP or dUDP -> dUMP + THF -> dTMP
Purine salvage pathways: what is the purpose
HGPRT is able to recycle purines so you don’t have to always make them from scratch
If they aren’t recycled they will end up as uric acid
Silent mutations
Nucleotide substitution codes for the SAME amino acid, usually has to do with change in the Wobble position
Transition mutation
purine to purine or pyrimidine to pyrimidine
Transversion mutation
purine to pyrimidine or pyrimidine to purine
Missense mutation
Nucleotide substitution resulting in changed AA
Nonsense mutation
Nucleotide substitution resulting in early STOP codon, usually creates a nonfunctional mutation
STOP THE NONSENSE
Frameshift mutation
Deletion/insertion that results in misreading of all nucleotides downstream
Protein can be shorter/longer, function may be altered
Splice site mutation
Mutation at a splice site that causes the INTRON to remain in the mRNA: causes protein to have impaired/altered function
Nucleotide excision repair: what is it? when does it occur?
Repairs bulky lesions that distort the helix, endonucleases remove sections of nucleotides that contain the bulky damage, DNAP and ligase fill in and seal the gap
Occurs during G1 phase of cell cycle
Base excision repair: what is it? when does it occur?
Occurs when a specific base was damaged: ex. due to spontaneous deamination, during any phase of cell cycle
- Base-specific glycosylase removes damaged base leaving a blank space
- Endonucleases come in and remove additional nucleotides at the 5’ end
- Lyase cleaves the 3’ end
- DNAP beta fills in gap
- Ligase seals gap
Mismatch repair: what is it? when does it occur?
Occurs when new strand base does not match with parent strand, usually during G2 phase of cell cycle
- New strand is recognized
- Mismatched nucleotides are removed
- Gap is filled and seals
Nonhomologous end joining
2 ends of DNA fragments brought together to repair ds-breaks
Start codons
AUG (CUG is rare alternative)
Stop codons
UGA, UAA, UAG
U Go Away
U Are Away
U Are Gone
PCR
Used to amplify a fragment of DNA
- Denature DNA by heating it
- Add primer that will bind to the sequence that you want to amplify
- Heat-stable DNAP replicates sequence after primer
Southern Blot
Used to ID a piece of DNA
- DNA sample is cleaved into smaller pieces that are separated on a gel by electrophoresis and then transferred to a filter
- Filter exposed to radiolabeled DNA probe that will bind to complementary strand
- Expose filter to film so you can see the DNA probe
“SNOW DROP”
Northern Blot
Used to ID RNA
Similar to southern blot except RNA
“SNOW DROP”
Western Blot
Used to ID a protein
Sample protein separated via gel electrophoresis and then transferred to a membrane
Labeled antibody is used to bind to the protein
“SNOW DROP”
Southwestern Blot
identifies DNA-binding proteins using labeled oligonucleotide probes
Flow cytometry
Used to assess size, granularity, protein expression of individual cells in a sample
Cells are tagged with specific antibodies, a laser focuses on the cell and then measures the intensity of the fluorescence
Data plotted on histogram or scatter plot
Microarrays
Nucleic acid sequences arranged in grids on glass or silicon
DNA or RNA probes are hybridized to a chip and a scanner is used to determine the amt of complementary binding
Used to profile gene expression of THOUSANDS of genes simultaneously to study certain diseases and treatments
Able to detect SNPs
ELISA
Immunologic test used to detect the presence of a specific antigen or antibody
You have antibody bound to an enzyme, then you add substrate that will react with the enzyme to produce a color
FISH: Fluorescence in situ hybridization
Fluorescent DNA/RNA probe binds to specific gene site of interest on chromosomes
Allows you to view microdeletions, translocations, duplications on chromosomes
Cloning method
- Isolate eukaryotic mRNA of interest
- Exposed mRNA to reverse transcriptase to produce cDNA which does not have introns
- Insert cDNA fragments into bacterial plasmids that contain antibiotic resistance genes
- transform the recombinant plasmid into bacteria
- Plate on antibiotic medium: the ones that survive have cloned copies of DNA in them
Microsatellite testing
Test for genetic differences btw people, looks at length of microsatellite repeats
Paternity test
