Download MCB 2050 midterm UPDATED ACTUAL and Correct Answers and more Exams Medicine in PDF only on Docsity!
MCB 2050 midterm UPDATED ACTUAL and Correct Answers
Question: Gene Cloning Steps
Answer:
- Digest vector and DNA of interest with same RE
Question: Anneal/ligate DNA to vectors forming recombinant plasmid
Answer:
Question: Transform plasmid -> E. coli
Answer:
Question: Selection (method of screening) then amplify
Answer:
Question: Restriction Enzymes (RE)
Answer:
- Cut at specific "restriction" sites in DNA (phosphodiester bond) (palindromic sequences) Function: degrade invading DNA (don't degrade if methylated)
- Cohesive/blunt ends
Question: 3 essential components of cloning, expression, and reporter, plasmids.
Answer: -origin of replication -dominant selectable marker (antibiotic resistant gene)
- Unique RE sites for gene insertion
Question: pBluescript
Answer: MCS in lacZ
- if inserted, nonfunctional = white
- if not inserted, functional = blue
Question: Eukaryotic expression vectors
Answer:
- Bacterial Ori --> propagate plasmid and selectable marker
- Eukaryotic promoter, MCS & polyadenylation signal (poly A tail) to express protein of interest in cell
- Eukaryotic selectable marker, to select cells with plasmid after transfection and drug selection
Question: Constructing DNA libraries
Answer:
- Cut DNA and vector with same RE
- anneal/ligate --> recombinant plasmid
- Plat on agar w/ drug selection Clone library: each colony has a plasmid with a fragment of DNA, representing a portion of DNA used to construct the library
Question: Constructing a cDNA library
Answer: -Isolate mRNA and convert to cDNA before making library
- Reverse Transcription and oligo dT primer = 1st strand -degrade mRNA w ribonuclease H
- synthesize 2 strand w DNA polymerase (from 1st)
- Screen library by complementation/hybridization
Question: Complementation
Answer: won't survive without glucose, so remove it to isolate
Question: Hybridization
Answer: radioactive probe complementary to sequence
Question: Electrophoresis
Answer: Size-based separation on gel matrix and electric field -all = -ve
Question: DNA restriction mapping
Question: Polymerase Chain Reaction (PCR)
Answer: -Forward and reverse primer (amplify each DNA strand) Steps: -Denature template DNA (95˚C)
- anneal primers to template (55˚C)
- extend primers w/ thermostable polymerase (taq, pfu) and dntp's (72˚C) -Repeat x
Question: Pro's/con's of PCR
Answer: Pros: -Rapid turnaround -Sensitivity (small amounts of organisms can't grow in lab can be detected) Cons: -sample contamination -Test inhibition (sample contains things that interfere with test) -Lack of validation (hard to prove for sure)
Question: DNA sequencing
Answer: Sanger method: every time a fluorescent ddntp incorporated, elongation stops, therefore you know that nucleotide -size of sequence determines color, can order from smallest to largest, will be complementary to templateDNA)
Question: SDS PAGE
Answer: -Separates proteins based on size -SDS denatures proteins and net -ve charge
Question: Western blotting
Answer: separate proteins on membrane for detection w specific antibody -1˚ antibody binds to protein of interest -2˚ antibody conjugated to an enzyme specific for 1˚ antibody
- substrate light via enzyme activity
- light measured by xRAY film, intensity proportional to amount of protein
Question: pros' cons of western
Answer: pros: -relatively fast -good to detect relative amount/size of protein cons: -not as good for relative abundance of a protein in a tissue
Question: Immunoflourescence imaging
Answer: -Antibody to detect protein localization in fixed cells
- 2˚ antibody taged w flourophore (visualize with flourescence microsope)
- fluorescent protein: GFP --> location in REAL TIME
Question: cytological mapping
Answer:
- banded pattern of stayed chromosomes
- G bands giemsa on mitotic chromosomes
- detect translocations
- In situ hibridization positions genes/gene probes on chromosomes
- FISH (fluorescence in situ hybridization) - biotin bound probes, detected with avidin bound to fluorescent dye
Question: Physical map
Answer:
- Molecular biology techniques arrange sequence features on linear map
- Location of DNA sequences on a chromosome
Question: Contig Map
Answer:
- Overlapping genomic DNA clones ordered by restriction/STS mapping or DNA sequencing -> sequence tag sites (STS): short/unique, (200-500 bp) derived under whole genome sequencing -> Expressed sequence tags (EST): short cDNA sequences Presence of particular STS/EST: order genomic clones relative to position of STS/EST
Question: Genetic Mapping
Answer:
Question: Human Genome project
Answer: to sequence and map human genome -Celera: privately owned, shotgun sequencing: randomly fragment genome, sequence each fragment and order with a computer
Question: Comparative genomics
Answer: genome evolution: compare genome b/w organisms -deduce structure/function of gene products -phlogenic trees
Question: transcriptome
Answer: The entire set of mRNA transcripts in a biological sample
Question: DNA microarray
Answer: measures gene expression (large scale) -many DNA probes attached to solid support (order) -flourescent cDNA hybridized microarray and bound cDNA --> info on which RNA expressed and relative abundance
Question: Comparative microarray
Answer: cDNA from 2 sources w 2 fluorescent dyes -uses transcriptome profiling, gentling (SNP) diagnostics
Question: Proteome
Answer: entire set of protein in biological sample comparative proteome: 2 samples
Question: Interactome
Answer: all protein-protein interactions in biol. sample
Question: Huntingtons Disease
Answer: Gain of function dominant mutation HTT -> mHTT
- HTT <35 CAG (glutamine/Q) repeats -mHTT >40 CAG repeats (misfolded = toxic aggregate) Diagnose: PCR -> size of CAG region
Question: Cystic Fibrosis
Answer: -Loss of function of CFTR, which codes for Cl- channel -F508 = most common mutation (F deletion), CFTR misfolds in golgi and is degraded, doesn't get to membrane Diagnose: PCR products w/ allele specific probe
Question: correcting mutant phenotype
Answer:
- add normal copy of gene (transgene)
- recombinent retrovirus = stable insertion, but insertion site is random
Question: ADA-SCID (adenosine deaminose deficient sever combined immunodeficient disease
Answer: -accumilation of deoxyadenosine in absence of ADA activity --> toxic to lymphocytes = death/immune deficiency
Question: X-linked SKID
Answer: -mutation in IL2R¥e fwnw = T cell death (lack of interleukin Z) --> wild type T IL-2 in retroviral vector from bone marrow/stem cell, grow in culture, return/implant back to patient
Question: Recombinant Protein Producition
Answer: -Recomb. plasmids express proteins of economic value in GE bacteria or Euk cells (insulin, GH, clotting factor IV) Bacteria: cost effective but lack post trans modificats -Yeast, insect/mamalian cell
Question: Generating TG animals by microinjecting DNA to fertilized eggs
- Thymidine kinase gene in target vector = select against cells that have random integration (not homologous) (cells killed by gancylovir = toxic when thymidine kinase (P)s it) -cells ` take up killed by neomycin
Question: T-DNA insertions
Answer: collect of TG plants w genes disrupted -> random tDNA insertions
Question: RNA interference
Answer: -dsRNA/short hairpin RNA can binds RNA 3' UTR and inhibit translation/cause mRNA degradation -RNAi: cell naturally pathway to silence gene expression (miRNA/RISC) -Technique used to silence expression of any gene w known sequence
Question: miRNA
Answer: small 1 strand cellular RNA to control gene expression
Question: RISC
Answer: RNA Induced Silencing Complex
Question: CRISPR/Cas
Answer: exploits natural defense system by using bacteria to guard against bacteriophage infection -single guide RNA (gRNA) targeting gene of interest is transfected to cells, w gene coding Cas protein
- Double stranded cut made in genome@ target site (repaired by DNA repair enzymes)
- non-homologous end joining repair = insertion/deletion, gene = inactive
Question: Multiple levels of control
Answer: Transcriptional -TF to dna sequence motif in promoter mRNA processing
-polyA tail, exons/introns (splicing) Translational control Post-translational control
Question: Signal transduction
Answer: extracellular signal to intracellular target = response
Question: Regulation by env. stress
Answer: heat shock TF HSF -trimerizes w proteotoxic stress
- binds to HSE present in promoter = enhances in HS protein genes -trimerization repressed in non-HS genes
Question: Steroid hormones
Answer:
- LS hormone diffuse to membrane to intracellular receptor which enters nucleus and brings to hormone response elements (HRE) -> enhanced in hormone regulated genes
Question: Peptide hormones
Answer: binds to Rm, activate signal transduction cascade, lead to TF activation (promoting a change)
Question: DNA sequences controlling gene expression
Answer: -Basal promotor elements (TATA, GC, CAAT) bind: --> general TATA proteins (general TF) --> basal TF (NF1, Sp1) --> Polymerase 2 -Enhancers: bind =activate TF -> specific regulation of gene expression i) act over large distances ii) orientation INDEPENDANT iii) position independent (up/downstream, intron)
Regulation of gene expression by changes in chromatin structure (heritable and reversible) -heterochromatin: densely packed, transcriptionally inert -Euchromatin: loosely packed, transcriptionally active -Position effect variation: occurs when gene transferred b/w euchromatin and heterochromatin regions, resulting in altered gene expression (drosophila mottled eye)
Question: chromatin remodeling
Answer: -histon acetyl transferases (HATs) --> acetylate histone lysine residues = losen DNA-nucleosome interaction -histone deacetylases (HDACs) remove acetyl group = compaction -Mating type switching (SWI)/sucrose non fermenting ATPases (SNF) --> shift nucleosome along DNA = clear promotor region for TF binding
Question: DNA methylation and gene imprinting
Answer: -DNA methyltransferases --> attach methyl to cysteine residue in CpG dinucleotides, recruiting chromatin modifying proteins which compact and inhibit gene expression -Imprinted genes --> methylation pattern = inherited (IgF2 gene = CH3 in F but not M) only copy from F is transcriptionally active
Question: Inactivation of whole chromosomes
Answer: mammals: females XX = X inactivation drosophila: XY male = X hyperactivation C elgans: XX hermaphorpdite = both hypoactive X's
Question: Alternate splicing of mRNA
Answer: mature mRNA differs even from same DNA
Question: Cytoplasmic control of mRNA stability
Answer:
-PABP1/elf-4E mediated circularization prevents mRNA decay -deadenylation = binding deadenylating enzyme = destabilize AUUUA in 3' UTR = short lived
Question: microRNA
Answer: -synthesized as pre-miRNA w ds hairpin structure -drosha removes ends --> pre miRNA exported to cytoplasm -Dicer removes hairpin loop -> 22 nucleotide dsRNA -dsRNA associates w AGO2 protein = RISC
- strand are unwound, guide base pairs w mRNA 3' UTR, other strand destroyed, results in mRNA silencing or degradation
Question: siRNA
Answer: small interfering RNA, defines against transposon
Question: miRNA
Answer: regulate gene expression
Question: molecular chaperone protein
Answer: regulate proteostasis -binds to exposed hydrophobic domains on unfolded protein preventing aggregates -assist in protein folding and intercellular transport -deliver damaged protein to UPS
Question: UPS (ubiquitin proteasome system)
Answer: damaged and short lived protein are ubiquitinated and delivered to proteasome for destruction
Question: Properties of tumor
Answer:
- mutation of critical genes for cell proliferation/death do to env. factors on epithelial cells
intrinsic: caspase 9 -> intracellular signal/ cell suicide extrinsic: caspase 8 -> death receptor/instructive apoptosis (cell murder)
Question: BCL-2 family proteins
Answer: regulate cytochrome c release from mitochondria -BAX: pro apoptotic, activation = channels in OMM to release cyt. c -- cyt c release = apoptosome formation (APAF1 digomerization) and caspase 9 activation -BCL2 = anti apoptotic, prevents BAX activation -BH3 = only protein that inhibits antiapoptotic members and directly activate BAX
Question: Oncogenes
Answer: promote cell proliferation and survival -> arise from point mutation, translocation, or gene amplification of proto-oncogenes
Question: Src
Answer: intracellular kinase controlling proliferation --> avian retrovirus C-src gene acquired during infection = V-SRC = trunkated or constitutively active
Question: Point mutations
Answer: Ras: monomeric G protein (GTP = active) transmits signal from receptor tyrosine kinases (RTK) which cause intracellular kinases (MAPK) controlling cell proliferation point mutation Ras^D: constitutively active
Question: point mutation/deletion
Answer: produce hyperactive receptors (EGFR), growth factor independent activation
Question: Gene amplification
Answer:
EGFR & C-MYC genes amplified = increased expression level -Burkits lymphoma: reciprocal translocation b/w causing c-myc gene under immunoglobin gene enhancer = overproduction of c-myc
Question: Tumor suppressor genes
Answer: inhibit proliferation or survival, point mutations/promoter methylation = loss of function mutation
Question: Retinoblastoma protein (Rb)
Answer: brake cell cycle by inhibiting TF E2F, phosphorylated pRb can't inhibit it (cyclin D (P) it) = hyperphosphorylated Rb)
Question: p53 (guardian of genome)
Answer: -TF kept at low levels by MDM2 ubiquitination and protease degradation --> p53 accumilates in stressed cells, ^ transcription of genes for DNA repair, cell cycle arrest (p21), and apoptosis (BAX) p53 mutated in 50% of cancers (tetrameric, so mutation= active), cells then escape apoptosis (tumorigenic mutation)
Question: Six essential cancer hallmarks
Answer:
- acquire self sufficiency in growth signalling (receptor mutation or mutation downstream from signalling proteins)
Question: insensitive to antigrowth signals (TGFB receptor/signal protein mutation)
Answer:
Question: evade apoptosis (overexpress anti-apoptotic/loss of function in pro-apoptotic)
Answer:
