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86 Cards in this Set
- Front
- Back
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Genome
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Complete set of information in an organism’s DNA that an organism will use.
Only a small portion of the human genome encodes protein-coding genes with the majority of sequence containing regulatory and repetitive regions. |
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Genomic Evolution
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Genome sequences of 2 species differ in proportion to the length of time they have separately evolved.
Sequences comparisons of different species genomes show how genomic structure has diverged, can lead to the identification of new for genes, and reveals important DNA sequence of unknown function. Gene duplications and divergence give rise to families of related genes with related function within a cell and play a role in the evolution of increased biological complexity. Genes are more conserved (especially protein-coding, RNA coding, and regulatory coding) than genomic structure overall. ⬆Genes = ⬆Complexity |
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DNA Structure
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Sense strand = 5'-3'.
Anti-sense strand = 3'-5'. DNA/Protein interactions occur in major groove |
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Purifying Selection
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Mutation has occurred, but organism can't survive and is thus selected against (unable to pass on genes)
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Synteny
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Stretches of conserved gene order on chromosomes
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New Gene Creation
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Genetic Variation Among Humans
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single nucleotide polymorphisms - these are positions in a genome where some individuals have one nucleotide and others have a different nucleotide.
simple sequence length polymorphisms - tandem repeat sequences that display length variations. (minisatellites - variable number tandem repeats (VNTRs), microsatellites - simple tandem repeats) |
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DNA Replication
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Semi-conservative (always have original copy). Strand elongation - 5'-3', requires primer, and free 3' OH.
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DNA Polymerase
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Adds bases 5'-3', Exonuclease activity - 3'-5'. Responsible for strand elongation
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Replication fork
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Leading and Lagging strand (Okazaki fragments) are coded. RNA primer is required for lagging strand.
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DNA Primase
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Adds RNA primer so lagging strand can be synth.
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RNAse H
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Removes RNA primer
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DNA Ligase
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Seals nicks.
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DNA Helicase
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Prys apart the double helix through the hydrolysis of ATP
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Single Strand DNA Binding Protein
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Prevents single strands from forming hairpins
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Sliding-Clamp Protein Complex
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Holds DNA poly on DNA template
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DNA Topoisomerase
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Prevents DNA tangling during replication.
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DNA Repair
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Type of repair depends on type of damage, stage of cell, and enzyme molecules involved in repair mechanism
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Base Excision Repair
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Nucleotide Excision Repair
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Recombinatorial Repair
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Strand-Directed Mismatch Repair
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DNA Recombination
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Large portions of DNA are involved, compared to mutations (base pairs) and cause more biological variability w/o sacrificing fidelity
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Homologous Recombination
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Occurs over long stretches of DNA in homologous chromosomes during meiosis.
Double stranded breaks allow for this to occur. Can occur w/ or w/o crossing over, crossing over causes Holliday-Junctions. Used as a form of DNA repair. |
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Site-Specific Recombination
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Movement of short specialized nucleotide sequence ,between non-homologous sites that can occur at anytime.
Short-inverted repeats allow transposon to insert. Transposons Not a form of DNA repair |
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Histones
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Pkg chromatin.
Octomeric Core (H2A, H2B, H3, H4). H1 expressed when tightly packed, results in silencing. Many variants, N-/C-terminals are varied based on fxn. Tails hang out to be modified (regulation). Histone wrapping determined by histone code (enzymatic modif. + sequence) that regulates availability → Amt. of expression. Epigenetic histone code can be inherited. |
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Chromatin structure and ATP-driven chromatin remodeling complex
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Dynamic and only short window for proteins to bind.
ATP driven chromatin remodeling complex - moves, ejects, or restructures nucleosomes (exchanges subunits) |
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HAT/HDAC and HMT/HDMT
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HATs – Histone acetyltransferases
HDACs – Histone deacetylases HMTs – Histone methyltransferases HDMTs – Histone demethylases Modify N-terminus of histone tail |
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Genetic Swtiches
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Transcriptional activators - turn on gene expression.
Transcriptional repressors - turn off gene expression. Co-activators/co-repressors - do not bind DNA but assemble on DNA bound regulatory proteins. Trp gene switch uses a ligand instead. |
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Combinatorial Gene Regulation
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Combination of gene switches can determine what cell is developed.
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Heterodimerize
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Different regulatory proteins coming together that recognize different sequences
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Major/Minor Groove
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Contain distinct pattern of H-bond acceptors/donors, methyl groups, and hydrophobic patches that regulatory proteins recog. (Helix-turn-helix, Zn-Finger proteins)
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Transcription vs Replication
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Both have template strand, 5'-3', use polymerase.
Transcription does not require a primer and can use either strand (promotor/histone modif.) determines which strand is used). Replication requires primer. |
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RNA
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mRNA - makes up transcriptome
rRNA - most abundant tRNA smRNA (snRNA - mRNA processing, snoRNA - rRNA processing, miRNA - post-transcriptional gene-silencing) |
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RNA Poly
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I - transcibes rRNA
II - transcribes mRNA and snRNA genes III - transcribes tRNA and other genes |
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Transcription Factors
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Recruit RNA poly to promotor, position and aid binding of RNA poly to promotor.
TF2D TBP- recog. TATA box which is part of promotor. TAF - recog. DNA seq. near transcription start point and regulates DNA binding by TBP. TF2H unwinds DNA at start point (helicase fxn) and phosphorylates Ser5 on CTD on RNA poly (impt. for post-transcript modifications). |
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Transcription Initiation
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TF2D binds first and recruits other TF and RNA Pol II to the transcription site.
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Transcription Enhancers
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Bind to site far away from transcription site. Loops back and binds to the transcription complex
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RNA Processing
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Occurs at the same time as transcription (only in eukaryotes). Involves splicing, capping, and poly-A.
Phosphorylation from TF2H attract capping/splicing factors/proteins (mutation in TF2H kinase ability = no RNA processing). 5' Cap is first. Poly-A tail is next. Cell knows RNA ends by sequence recog (A/U regions preceding a CA that marks cleavage site) and A's added to CA. Amount (length of A) determines how long RNA is present and avail. (more = more stable) |
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RNA Splicing
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Alternative splicing can lead affect expression. AG/GU and cell type direct alternative splicing.
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Pre-mRNA Splicing Pathway (Part 1)
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U1 recognizes/binds AG/GU site.
U2 binding causes A to bulge out. U4/U5/U6/ stabilizes |
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Pre-mRNA Splicing Pathway (Part 2)
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U6 stabilizes 3'-OH
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Pre-mRNA Splicing Pathway (Summary)
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Modes of Alternative Splicing
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Yellow = exons.
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Regulation of Alternative Splicing
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Export of mRNA
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Need Poly/Cap to leave nucleus
CBC – cap binding protein – protects cap EJC – exon junction proteins Poly-A binding proteins – protect poly a SR- serine/arg proteins – involved in splicing and will fall off before going to translation Nuclear pore recognizes cap and allows mRNA to leave |
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mRNA Stability and Degredation
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5' cap and length of poly-a tail are what determine stability.
Degradation can occur in nucleus and cytosol. Degradation mediated by Deadenylation nuclease (DAN) - associates w/5' cap of mRNA and shortens the poly-A tail in the 3'→5' |
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miRNA Mediated Gene-Silencing
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21-23 nucleotides, silences genes, often processed in clusters, form hairpin loops 200-300bp
a.) DROSHA processes Pri-miRNA→Pre-miRNA in nucleus b.)Exportin5 uses GTP to transport Pre-miRNA out of nucleus c.) DICER cleaves hairpin loop leaving miRNA duplex (more stable) in cytosol d.) Duplex unwound and incorporated into RISC. e.) RISC complex binds mRNA. completely complementary - cleavage mismatch - translation inhibition |
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Ribosome
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80S
60S - 5S, 28S, 5.8S, 49 proteins 40S - 18S, 33 proteins A site - amino acyl tRNA site P site - peptidyl site E site - exiting site Start codon (aka Kozak sequence) sets the reading frame. |
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Aminoacyl-tRNA synthetase
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2-Step
1.) Linkage of specific amino acid to a specific tRNA (ATP dependent) 2.) tRNA binds to the codon Hydrolytic Editing Occurs in tRNA synthetase. Excludes aa that are too big, aa hydrolyzed off tRNA |
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Translation Initiation
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Repressors can hide the start sequence; miRNA also involved.
eIF (Eukaryotic initiation factors) 4 binds mRNA at 5' cap and 3' poly-a tail and mRNA is positioned in the small ribosomal subunit. eIF-2 associates w/tRNA containing Met eiF-2 dissociates via hydrolysis of GTP, large ribosomal subunit binds. |
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Translation Elongation
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EF (elongation factors) enhance translation and control accuracy
EF-1 proofreads - tells if tRNA has right aa and is bound to right codon EF-2 placeholder - binds A site until ribosome ready for new tRNA Hydrolysis of GTP has to occur for ribosome to shift to next codon. New peptide bond is formed at P site. |
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Translation Termination
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Release factor binds to A site with stop codon, causes termination.
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Antibiotics
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Actinomycin D - binds to DNA and blocks movement of RNA Poly → preventing RNA synth
Cyclohexamide - blocks translocation reaction on ribosomes Tetracycline - blocks binding of aminacyl-tRNA to A-site of ribosome |
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Post-translation Control
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Reversible - folding, addition of functional groups, structural changes, processing, removal of start methionine
Irreversible - proteolysis Some proteins start to fold as they are being translated. Not all proteins require a chaperone to fold. |
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Compartmentalization
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Protein processed at various areas in the cell while being transported to its target.
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Epigenetic Control of Gene Expression
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Regulation of histone modification and heterochromatin - transcriptionally activated/silenced, modified histones ensure that differentiated cell would stay that way, most epigenetic changes only occur w/in course of organism's lifetime, but can be inherited. miRNA target HAT/HDAC
DNA methylation - ⬆Methylated areas are transcriptionally less active (CPG islands). DNA methylation patterns are inherited. DNA methyltransferases (DNMT's) - localizes to replication fork and transfers patterns of methylation to newly synthesized DNA after replication; essential in embryonic development, imprinting, and x-inactivation. Genomic Imprinting Gene Silencing X-Chromosome Inactivation |
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Cleavage of DNA w/ Restriction Endonucleases
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Digestion of phosphodiester backbone
Palindrome 5' overhang (sticky) 3' overhang (sticky) blunt |
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Gel Electrophoresis
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Separation by MW (size)
Agarose and polyacrylamide DNA has net negative charge → travels to anode (+), can see single bp differences DNA made visible by radioactive isotope, EtBr, etc. |
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DNA Cloning
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use of vectors makes it possible
- ds circular molecules - occur naturally in bacterial cells - self-replicating - small in size |
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Plasmid Vectors Features
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- OriR
- MCS containing restriction sites to facilitate cloning of insert DNA - antibiotic resistance gene - allows for selection - small size - efficient transformation - LacZ gene - allows for selection of recombinants (clones are white if gene is disrupted) |
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Transfornation
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DNA into bacterial cell
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Transduction
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Virus into mammalian cell
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Transfection
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DNA into mammalian cell
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cDNA library
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Tissue → lysed cells → get mRNA → RT to get DNA of 1 strand, then DNA pol to replace mRNA template → Digest → DNA fragments
Allows you to see what was expressed/coded |
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Genomic DNA library
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Digest entire genome → DNA fragments
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Nucleic Acid Hybridization
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DNA denatured and renatured. Complimentary single strands of DNA readily reform into dsHelices
Can occur between DNA-DNA, RNA-RNA, DNA-RNA. Probes must be ss, and can be labeled w/radioisotopes, fluorescent dyes. Able to detect how many copies of DNA/RNA are in a cell and can detect differences in a single bp. Changing stringency (hybridization temp can affect tightness of binding = specificity) |
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In-Situ Hybridization
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Occurs in an intact cell, tissue, or chromosome
Reveals dist. of a particular DNA or RNA molecule w/in a cell or location of a gene on a chromosome |
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Northern/Southern Blotting
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Northern = RNA
Southern = DNA Detects - Gene of interest - Genes that are related but not identical - Certain RNA transcript being processed - Size, abundance of RNA transcript - Map position of introns/exons as well as start/stop sites for RNA transcription - Single nucleotide differences and genetic diseases |
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Microarray Analysis
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Examine expression of many genes in one experiment
Glass slides containing DNA probes that identify each gene (each spot) DNA/RNA extracted fro cells and is labeled w/fluorescent marker Uses - Able to study thousands of genes at the same time - Screening and diagnostic tool - Drug/therapy development - Expression patterns/levels |
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PCR
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Ingredients
Template DNA Gene specific primers (probes) dNTPs Taq (DNA) Poly favorable rxn conditions Steps Denaturation (94C) Annealing (Variable - depends on primer G/C content, length, and mismatch) Extension (72C) Amount dNTP is limiting reagent Uses Carrier screening Pre natal diagnostic testing Newborn screening Confirmatory diagnosis of symptomatic individ. Clone genomic DNA and cDNA |
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Quantitative RT-PCR
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Can be combined w/RT to quantify mRNA in cells/tissues.
(mRNA + RT = cDNA) Allows detection and quantification - absolute number of copies - relative amt of copies when normalized to DNA or normalizing genes Quantify Using fluorescent dyes that intercalate w/dsDNA Modified DNA probes that fluoresce when hybridized w/cDNA |
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DNA Sequencing
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Uses DNA poly
- requirements are template, primer, free 3'-OH - synthesis of new DNA in 5'-3' - ddNTP used, stops polymerization (no free 3'-OH) = results in lots of fragments Shotgun sequencing Automated Break up the genomic DNA into fragments - determines the sequence of each fragment - use computer programs to search for overlap |
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Genetic Mapping
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Using genetic techniques to construct maps showing positions of genes and other DNA markers on a genome
Based on principles of inheritance and genetic linkage. |
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DNA Markers
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SNP
SSLP (simple sequence length polymorphisms) - repeat sequences that display length variations - minisatellites or variable number tandem repeats (VNTR) - microsatellites or simple tandem repeats RFLP |
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RFLP
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Polymorphic restriction sites, existing as two alleles
One allele displays correct sequence for restriction site and second has sequence alteration (restriction site not recog) Able to indicate presence of disease locus |
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Physical Mapping
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Using molec. bio techniques to examine DNA molecules directly to construct maps showing postions of sequence features, including genes
Restriction mapping - positions of recog. seq. for restriction enzymes FISH - position of gene or DNA marker on chromo to be directly visualized w/probe STS mapping - positions of unique sequences that occur only once in the chromosome of genome being studied |
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Expression Strategies (Bact. vs Mamm)
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Bacterial Expression
- Cloned cDNA, promotor must be present in plasmid to control expression of gene, no post-translat. modif. Mammalian Expression - Cloned cDNA, into cancer derived cells, mammalian promoter myst be present in plasmid and antibiotic selection, post. translat. modif. |
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Reporter Molecules and Epitope Tags
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Reporter molecule (gfp)
- gene is cloned w/epitope tag and transcribed/translated → ab hybridized to gfp recognizes epitope tag on protein → paints the protein Epitope tag (GST) - allows you to use reporter molecules and examine cell-protein interactions - gene is cloned w/epitope tag and transcribed/translated → "GST purification from undergrad, except proteins that interact w/tagged protein aggregate on it Yeast-2 hybrid BAIT/PREY - BAIT has DNA binding domain and is coded w/target protein - PREY has transcript. act. domain and is coded w/binding partner - Both bind in cell and activate transcription and expression of reporter protein. |
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FRET
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Lets you see protein localization and interactions
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Site-Directed Mutagenesis
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- Primer w/single bp mismatch is used to modify protein coding sequence
- Employ annealing strategies to allow single bp mismatch - Mutation gets incorporated and get protein w/ altered fxn |
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Anti-sense Technology
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Short DNA/RNA ssOligonucleotides that are complementary to sp gene structure → Inhibits gene expression
Blocks by - blocking RNA splicing and initiation of translation - prevent transport of mRNA antisense complex into cytoplasm - increases RNA degradation |
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siRNA
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Same mechanism of interference as miRNA
Extensive match = mRNA degredation Less Extensive match = reduced translation |
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Embryonic Stem Cell Technology
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One way of producing transgenic mice
Target gene in embryonic stem cells (ES) - Based on homologous recombination (see photo) Select for cells expressing gene (neoR and HSV-tk) Inject transformed ES into inner cell mass of blastocyst Implant in uterus in foster mother Test offspring for presence of gene Profit |
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Cre/Lox Technology
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Form of conditional gene targeting
LoxP is the recognition site for Cre recombinase enzyme. LoxP sites are placed flanking target gene. This allows gene to be eliminated under certain conditions (tissue type/time) Spatial = Global Cre expression vs. tissue specific Temporal = Inducible Cre expression w/genetic on/off switch (tet/estrogen) |
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Pronucleus Micro-injection method
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Inject gene into pronucleus.
Rate of Non-homologous recomb is high and gene is integrated Implant egg into foster mother Test offspring for presence of gene Profit |
