DNA polymerase used in eukaryotes for DNA replication
alpha/delta DNA polymerase
Characteristics of DNA replication in the leading strand
Continuous and toward replication fork
Characteristics of DNA replication in the lagging strand
Starts from Okasaki fragments and directed away from replication fork
Main genes involved in mismatch repair
hMLH1 and hMSH2
Lynch syndrome confers an increased risk for developing which cancers
- Colon (HNPCC)
- Endometrial
- Ovarian
- Gastric
Mismatch repair occurs during which phase of the cell cycle
G2
Subunits of prokaryotic ribosome
30S + 50S = 70S
Subunits of eukaryotic ribosome
40S + 60S = 80S
Antibiotics that bind to the 30S subunits
“buy AT 30, CCEL at 50”
- Aminoglycosides
- Tetracyclins
Specific mechanism of action of aminoglycosides
Binding to the 30S subunit and inhibit the formation of the initiation complex / they also cause misreading of mRNA
Specific mechanism of action of tetracyclins
Binding to the 30S subunit and inhibit tRNA binding to the A-site
Antibiotics that bind to the 50S subunits
“buy AT 30, CCEL at 50”
- Cloramphenicol
- Clindamycin
- Eryhtromycin (Macrolides)
- Linezolid
Specific mechanism of action of macrolides
Binding to the 50S subunit and inhibit translocation of ribosome during elongation phase
*Same as clindamycin
Specific mechanism of action of clindamycin
Binding to the 50S subunit and inhibit translocation of ribosome during elongation phase
*Same as macrolides
Specific mechanism of action of chloramphenicol
Inhibits peptidyl transferase in the 50S subunit, leading to inhibition of protein chain elongation
Specific mechanism of action of linezolid
Binding to the 50S subunit and inhibit the formation of the initiation complex
Mode of inheritance of Chédiak-Higashi syndrome
Autosomal recessive
Defective gene in Chédiak-Higashi syndrome
Lysosomal trafficking regulator gene (LYST)
Pathogenesis of Chédiak-Higashi syndrome
There’s a microtubule polymerization defect leading to impaired function of phagosome-lysosome fusion
Signs and symptoms of Chédiak-Higashi syndrome
- Neutropenia
- Recurrent pyogenic infections (staphylococci and streptococci)
- Partial albinism
- Peripheral neuropathy
Laboratory findings in Chédiak-Higashi syndrome
- Giant granules in granulocytes and platelets
- Pancytopenia
- Mild coagulation defects
Functions of cilia
- Mucociliary clearance
* Motility of sperm cells
How do mismatch repair enzymes distinguish between old and new strands in prokaryotes
Template strand’s cytosine and adenine are methylated during DNA replication
What does semi-conservative replication refers to
To the use of a template strand for the synthesis of a new strand in a complementary and antiparallel fashion
Enzyme that excises RNA primer in eukaryotes
RNAse H
Signs and symptoms of xeroderma pigmentosum
- Extreme sensitivity to sunlight
- Skin freckling
- Skin cancer early in life
- Ulcerations
Laboratory diagnosis of xeroderma pigmentosum
Measuring excision endonuclease levels in serum white blood cells
Treatment of xeroderma pigmentosum
Avoid UV light exposure
Compounds that can cause base deamination
Nitrates
*Found in meat preservatives (hot dogs!)
Consequence of a mutation in hMLH1 and hMSH2
Microsatellite instability
What are microsatellites
Repeating sequences of noncoding DNA
RNA processing modification that occurs co-transcriptionally (while mRNA is being transcribed)
Capping of the 5’ end (addition of 7-methylguanosine cap)
RNA processing modifications that occur post-transcriptionally (after mRNA has being transcribed)
- Polyadenylation of 3’ end (around 200 A’s)
* Splicing out of introns
Function of tRNA
Carries activated aminoacid for translation
Enzyme that catalyzes aminoacid activation
Aminoacyl-tRNA synthetase
Where does translation occur
Cytoplasm
Where in mRNA does the 40S subunit (eukaryotes) bind to in the initiation phase of protein synthesis
It binds to the 5’ cap structure
Where in mRNA does the 30S subunit (prokaryotes) bind to in the initiation phase of protein synthesis
Shine-Delgarno sequence
Ribosomal site where tRNA initially binds
Peptidyl site (P site)
Ribosomal site where the incoming tRNAs bind
Aminoacyl site (A site)
Mechanism by which shiga toxin inhibits protein synthesis
Cleaves an aminoacid from the 28S RNA of the 60S subunit of the ribosome
*Inhibits binding of tRNA to the A site
Enzyme that catalyzes the formation of the peptide bond inside the ribosome in protein synthesis
Peptidyl transferase
Part of the elongation phase of protein synthesis in which eEF-2 is required
Translocation of the growing peptide from the A site to the P site
Mechanism by which Pseudomonas and diphteria toxin inhibit protein synthesis
ADP-ribosylation of eEF-2
Cellular organelle affected in X-linked adrenoleukodystrophy
Peroxisomes
Trinucleotide repeat expansions are generated during which phase of the cell cycle
S phase
Types of mutation that a tumor suppressor gene needs to undergo to contribute to cancer
Deletions or los-of-function mutations
CDK2NA mutations can be found in what pathology
Melanoma
Protein products of CDK2NA gene
2 tumor suppressor genes:
- p16 protein is an inhibitor of cyclin D-Cdk4/6 complexes, causing the cell to stop at G1
- p14 protein inhibits MDM2, which normally targets p53 for degradation
Laboratory technique used in paternity testing to analyze repeated units of 2 to 6 base pairs
PCR to amplify the microsatellite repeats
*Microsatellite testing detects genetic differences between individuals
Laboratory technique that uses PCR to detect mutations occuring at sites where restriction enzymes cut
Restriction fragment length polymorphisms (RFLP)
What is the difference between 10 nm and 30 nm chromatin
The prescence of H1
Molecule needed by the prokaryotic RNA polymerase to start transcription
Protein factor sigma
Molecule needed by the prokaryotic RNA polymerase to end trasncription
Protein factor rho
Process inhibited by actinomycin D
Transcription, by inhibitng RNA polymerase in both eukaryotes and prokaryotes
Lecture direction of RNA polymerase
3’ to 5’ (reads the template strand)
Direction of synthesis of RNA polymerase
5’ to 3’
*The mRNA sequence will be identical to the DNA of the coding strand (uracil instead of thymine)
What is the primary level of protein structure
The sequence of aminoacids
What is the secondary level of protein structure
The folding of the aminoacid chain into energetically stable structures (alpha helix and beta sheets)
What is the tertiary level of protein structure
Positioning of secondary structures in relation to each other to generate 3 dimentional shape
What is the quaternary level of protein structure
The interaction among multiple subunits in proteins
Types of proteins translated in the RER
- Secreted proteins
- Membrane-bound proteins
- Lysosomal enzymes
Types of proteins translated in the cytoplasm (free ribosomes)
- Cytoplasmic proteins
* Mitochondrial proteins
Aminoacid sequence that when translated directs the nascent protein to the RER for the rest of its translation
Hydrophobic aminoacid sequence (branched-chain aminoacids)
*Ribosome stays attached to the RER
Name the 2 domains found in transcription factors
- DNA binding domains
2. Activation domains
Name the 3 types (and examples) of DNA binding domains in transcription factors
- Zinc fingers (eg, steroid hormone receptors)
- Leucine zippers (eg, cAMP-dependent transcription factors)
- Hélix-turn-helix (eg, embryonic gene expression transcription factors)
Function of the activation domain of transcription factors
- Binding to other transcription factors
- Interaction with RNA polymerase
- Recruitment of chromatin modifying proteins
Name and describe the types of transcription factors
- General: these are found in most genes within most cells, and are involved in basal transcription of genes
- Specific: localized to specific cells and tissues, they bind to specific enhancer/silencer regions modulating the formation of the initiation complex
Name the response element, function, and protein class of steroid receptors (transcription factors)
- Response element: HRE
- Function: steroid response
- Protein class: zinc finger
Name the response element, function, and protein class of cAMP response element binding (CREB) proteins (transcription factors)
- Response element: CRE
- Function: response to cAMP
- Protein class: leucine zipper
Name the response element, function, and protein class of peroxisome proliferator activated receptors (PPARs) (transcription factors)
- Response element: PPREs
- Function: regulate multiple aspects of lipid metabolism
- Protein class: zinc fingers
Name the response element, function, and protein class of NFkB transcription factor
- Response element: kB elements
- Function: regulate expression of many genes in immune system
- Protein class: rel domains
Name the response element, function, and protein class of homeodomain proteins (transcription factors)
- Response element: ?
- Function: regulate gene expression during development
- Protein class: helix-turn-helix
Definition of recombinant DNA
Splicing together of a human gene into a bacterial plasmid vector
Uses of recombinant DNA
- Provides a means of analyzing and altering genes and proteins
- Provides reagents for genetic testing
- Provides a source of specific proteins (eg, insulin)
Type of DNA sequence where restriction endonucleases cut
Palindrome sequences
*Yielding restriction fragments of chromosomes
Source of DNA in genomic libraries
Chromosomal DNA
Source of DNA in expression (cDNA) libraries
mRNA
Enzymes used to make genomic libraries
- Restriction endonuclease
* DNA ligase
Enzymes used to make expression (cDNA) libraries
- Reverse transcriptase
* DNA ligase
Main advantage of genomic libraries
When a non-coding sequence of a gene (like promoters or enhancers) needs to be studied
Main disadvantage of genomic libraries
That the gene of interest may be fragmented by endonuclease treatment
Main advantage of expression (cDNA) libraries
That the entire reading sequence of a particular gene is obtained intact, and therefore can be used in gene therapy
Vectors used in gene therapy to deliver the gene sequence to the somatic cell
- Modified virus
* Liposome
Will the gene delivered to the cells be inherited in gene therapy
No, because it is in a somatic cell