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MCB 2050 midterm Questions With Complete Solutions 3 essential components of cloning, expression, and reporter, plasmids. Correct Answers -origin of replication -dominant selectable marker (antibiotic resistant gene)
- Unique RE sites for gene insertion ADA-SCID (adenosine deaminose deficient sever combined immunodeficient disease Correct Answers -accumilation of deoxyadenosine in absence of ADA activity --> toxic to lymphocytes = death/immune deficiency Alternate splicing of mRNA Correct Answers mature mRNA differs even from same DNA apoptosis regulation Correct Answers controlled cell destruction by caspases (cytosine proteases that cleave after ASP residue) BCL-2 family proteins Correct Answers regulate cytochrome c release from mitochondria -BAX: pro apoptotic, activation = channels in OMM to release cyt. c -- cyt c release = apoptosome formation (APAF digomerization) and caspase 9 activation -BCL2 = anti apoptotic, prevents BAX activation -BH3 = only protein that inhibits antiapoptotic members and directly activate BAX
Cell cycle regulation Correct Answers -controled by specific cyclin dependent kinases, abundance of each kinase changes throughout cycle, -cancerous cells lose checkpoints CHIP Correct Answers -binding of TF to specific chromatin location in vivo (organism) -cells treated w. formaldyhyde = cross link protein and DNA -DNA sheared and mixed with antibody for specific TF -DNA/TF/Ab purified, protein removed, sequence isolated chromatin remodeling Correct Answers -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 Chromosome rearrangement Correct Answers -philadelphia: reciprocal translocation b/w 9/22 create fusion protein bcr-abl with constitutive tyrosine kinase activity = chronic mylenogenous leukaemia (CML)
Constructing DNA libraries Correct Answers - 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 Contig Map Correct Answers - 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 correcting mutant phenotype Correct Answers - add normal copy of gene (transgene)
- recombinent retrovirus = stable insertion, but insertion site is random CRISPR/Cas9 Correct Answers 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 Cas9 protein
- Double stranded cut made in genome@ target site (repaired by DNA repair enzymes)
- non-homologous end joining repair = insertion/deletion, gene = inactive Cystic Fibrosis Correct Answers -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 cytological mapping Correct Answers - 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 Cytoplasmic control of mRNA stability Correct Answers - PABP1/elf-4E mediated circularization prevents mRNA decay -deadenylation = binding deadenylating enzyme = destabilize AUUUA in 3' UTR = short lived DNA damage checkpoint Correct Answers p53 -> p21 -| CDK2/cyclinE/A -p53 = inhibitor protein, loss of function of p53 or of pRB (tumer suppressor gene)
iii) position independent (up/downstream, intron) DNA sequencing: Sanger method Correct Answers 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) Effector caspases Correct Answers caspase 3: = cleaved/activated by initiator caspases Electrophoresis Correct Answers Size-based separation on gel matrix and electric field -all = -ve
- shorter = faster Epigenetics Correct Answers 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) Eukaryotic expression vectors Correct Answers - 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 G1/S checkpoint Correct Answers GF synthesis -> cyclin D -| protein pRb -| E2F -> Cyclin E cyclin E drives from G1-> S Gel mobility shift assay Correct Answers radioactive DNA probe w binding site for a TF mixed w cell extract, separated on non-denaturing gel -DNA probe bound to TF = shifted in mobility during separation, demonstrating presence of that TF Gene amplification Correct Answers EGFR & C-MYC genes amplified = increased expression level Gene Cloning Steps Correct Answers 1) Digest vector and DNA of interest with same RE
- Anneal/ligate DNA to vectors forming recombinant plasmid
- Transform plasmid -> E. coli
- Selection (method of screening) then amplify Gene knockouts in mice Correct Answers -target vector containing neomycin resistance gene: inserting disrupts coding sequence of gene of interest
- ends of gene of interest in vector = homology with wilde type (recombination replaces genomic copy with disrupted copy)
Huntingtons Disease Correct Answers 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 Hybridization Correct Answers radioactive probe complementary to sequence Immunoflourescence imaging Correct Answers -Antibody to detect protein localization in fixed cells
- 2˚ antibody taged w flourophore (visualize with flourescence microsope)
- fluorescent protein: GFP --> location in REAL TIME Inactivation of whole chromosomes Correct Answers mammals: females XX = X inactivation drosophila: XY male = X hyperactivation C elgans: XX hermaphorpdite = both hypoactive X's initiator caspases Correct Answers intrinsic: caspase 9 -> intracellular signal/ cell suicide extrinsic: caspase 8 -> death receptor/instructive apoptosis (cell murder) Interactome Correct Answers all protein-protein interactions in biol. sample
Malignant cells Correct Answers spread to other tissues Methods to study regulation of gene expression Correct Answers -Reporter plasmid -Gel mobility shift assay -chromatin immunoprecipitation ChIP microRNA Correct Answers -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 miRNA Correct Answers regulate gene expression miRNA Correct Answers small 1 strand cellular RNA to control gene expression molecular chaperone protein Correct Answers regulate proteostasis -binds to exposed hydrophobic domains on unfolded protein preventing aggregates -assist in protein folding and intercellular transport -deliver damaged protein to UPS Multiple levels of control Correct Answers Transcriptional -TF to dna sequence motif in promoter mRNA processing
Physical map Correct Answers - Molecular biology techniques arrange sequence features on linear map
- Location of DNA sequences on a chromosome point mutation/deletion Correct Answers produce hyperactive receptors (EGFR), growth factor independent activation Point mutations: Ras Correct Answers 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 Polymerase Chain Reaction (PCR) Correct Answers -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 Position cloning Correct Answers Identif chromosoal position responsible for disease by association (pedigree), and with genetic markers (RFLP, STR, SNP) Pro's and cons of northern blotting Correct Answers pros: -relatively fast, low tech, and cheap
Cons: -less sensitive than PCR (100,000 copies to detect) Pro's/con's of PCR Correct Answers 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) Properties of cancer cells Correct Answers -deregulated cell division and death -anchorage indépendant growth -immortal -aneuploidy Properties of tumor Correct Answers - mutation of critical genes for cell proliferation/death do to env. factors on epithelial cells Pros and cons of southern blotting Correct Answers pros: -effective to detect specific DNA in long complex DNA -quatify amount of DNA present -Definitive to detect GMO, and cheaper than DNA sequencing Cons: -More expensive than most tests
Recombinant Protein Producition Correct Answers -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 Regulation by env. stress Correct Answers 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 Reporter plasmid Correct Answers sequential/targeted deletions to identify promoter sequences necesarry for transcript regulation Restriction Enzymes (RE) Correct Answers - Cut at specific "restriction" sites in DNA (phosphodiester bond) (palindromic sequences) Function: degrade invading DNA (don't degrade if methylated)
- Cohesive/blunt ends Restriction Fragment Polymorphisms (RFLP) Correct Answers Occur bc of an addition/deletion of RE site -Minisatellites (variable number tandem repeats VNTR) -> polymorphic locus 10-80 bp sequence in several tandem repeats
- Microsatellite (short tandem repeats STR) -> same as VNTR but shorter (<10 nucleotides)
Polymorphisms due to variable # of repeats b/w RE sites Retinoblastoma protein (Rb) Correct Answers brake cell cycle by inhibiting TF E2F, phosphorylated pRb can't inhibit it (cyclin D (P) it) = hyperphosphorylated Rb) Reverse Genetics Correct Answers Dissecting biological processes by inhibiting gene expression RISC Correct Answers RNA Induced Silencing Complex RNA interference Correct Answers -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 SDS PAGE Correct Answers -Separates proteins based on size -SDS denatures proteins and net -ve charge Signal transduction Correct Answers extracellular signal to intracellular target = response Single nucleotide polymorphism (SNP) Correct Answers 1 bp substitution in reference sequence --> Haplotype: set of SNP on same chromosome, inherited together
uses: detect homologous sequences, disease diagnostics, RFLP analysis (restriction fragment length polymorphism) Src Correct Answers intracellular kinase controlling proliferation --> avian retrovirus C-src gene acquired during infection = V-SRC = trunkated or constitutively active Steroid hormones Correct Answers - LS hormone diffuse to membrane to intracellular receptor which enters nucleus and brings to hormone response elements (HRE) -> enhanced in hormone regulated genes T-DNA insertions Correct Answers collect of TG plants w genes disrupted -> random tDNA insertions TG animals by ES cell technology Correct Answers embryonic stem cells = pluripotent stem cells from inner cell mass of blastocyst -mate mice and remove blastocyst from pregnant female
- isolate ES cells from blastocyst and culture in vitro
- Transfect transgene to ES cells -Inject transfected cells to freshly isolated blastocyst -implant blastocyst to pre female Screening: easier if black donor ES and white blastocysts -> offspring = chimeric, mate offspring, black MIGHT be transgenic TG plants from recombinant Ti plasmids Correct Answers -Ti plasmid of A. tumefaciens
- T-DNA region transfered to plant cell genome carrying genes required for plant cell transformation -Recomb. Ti plasmid replaces tumor gene w transgene (w selectable marker)
- plasmid inserted to A tumefacients, infect plant cells in vitro = TG plants EG. glyphosate resistant: mutant EPSP synthase transcriptome Correct Answers The entire set of mRNA transcripts in a biological sample Tumor suppressor genes Correct Answers inhibit proliferation or survival, point mutations/promoter methylation = loss of function mutation UPS (ubiquitin proteasome system) Correct Answers damaged and short lived protein are ubiquitinated and delivered to proteasome for destruction Western blotting Correct Answers 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 X-linked SKID Correct Answers -mutation in IL2R¥e fwnw = T cell death (lack of interleukin Z)
