Ch. 21: Structure & Properties Of DNA Flashcards
What does the primary structure of DNA consist of?
1) 5C deoxyribose sugar
2) Phosphate group attached to 5’ C of deoxyribose
3) Heterocylic nitrogenous base attached to 1’ C of deoxyribose
Which DNA bases are purines?
Adenine and Guanine
Which DNA bases are pyrimidines?
Cytosine, Thymine, Uracil
What does the secondary structure of DNA consist of?
Base pairing ➡️ overall architecture of DNA double helix
What does the tertiary structure of DNA consist of?
Superhelical structure (supercoil)
What are the 4 major stages that represent the central dogma of molecular biology? (DNA ➡️ RNA ➡️ pro)
1) DNA replication
2) Transcription: DNA codes for mRNA
3) mRNA processed (splicing)
4) Translation: mRNA brings info to ribosomes ➡️ used for pro synthesis
What were the 4 experimental outcomes (with mice) in the 1928 F. Griffith experiment?
1) Living S strain (virulent) put in 🐭 ➡️ 🐭 dies (S strain in tissue)
2) Living R strain (avirulent) put in 🐭 ➡️ 🐭 lives
3) Heat-killed S put in 🐭 ➡️ 🐭 lives
4) Mix of heat-killed S & living R put in 🐭➡️ 🐭 dies (S strain in tissue)
What were the 3 experimental outcomes in the 1944 Oswald Theodore Avery experiment?
1) Heat-killed S strain + RNAase + R strain (avirulent) ➡️ mix of R + S
2) Heat-killed S + protease + R ➡️ mix of R & S
3) Heat-killed S + DNase + R ➡️ only R
What is the transforming principle Oswald Avery & co discovered, in regards to the Griffith experiment?
DNA is agent responsible for genetic transferring (NOT pro!)
explains 4th outcome of experiment: turning avirulent R strain into virulent S strain via S DNA ➡️ kill 🐭
What were the 2 experimental outcomes of the 1952 Alfred Hershey & Martha Chase experiment?
1) bacteriophage pro coat radioactively labeled ➡️ pro coat remain outside bacterial host cell ➡️ not in pellet of bacterial cells ➡️ conclude: pro is NOT the genetic material directing production of new bacteriophages
2) bacteriophage DNA radioactively labeled ➡️ DNA injected into bacterial host cell ➡️ in pellet of bacterial cells ➡️ conclude: DNA is the genetic material directing production of new bacteriophages
Who discovered the double helical structure of DNA in 1953?
James Watson & Francis Crick
What is the difference between a nucleoside & nucleotide?
Nucleoside: Base + deoxyribose sugar
Nucleotide: Base + phosphate + deoxyribose sugar
What type of bond connects bases to form polymerized DNA strands?
Phosphodiester bonds: 3’ C of one sugar linked to 5’ C of next sugar
How is uracil obtained from cytosine?
How is thymine obtained from uracil?
Deamination of C ➡️ U
Methylation of U ➡️ T
Which DNA base pairs with which? (Chargaff’s rules)
How many H bonds are involved with each pairing?
A to T (2 H bonds)
G to C (3 H bonds)
Which are the most frequently methylated DNA bases?
Guanine
Cytosine (methylation pattern inherited)
What does DAM methylase act on?
Adenine in any GATC sequence
What does DEM methylase act on?
Cytosine in CCAGG sequence
Why is methylation important in bacteria?
Causes inactivation of DNA expression, protects against restriction endonucleases
What is genomic imprinting?
Different methylation pattern in maternal & paternal chromosomes at CpG nucleotides
What diseases involve faulty imprinting?
Prader-Willinsyndrome
Angelmann’s syndrome
Beckwith-Wiedemann syndrome
Example of good DNA methylation?
Women born with 2 X chromosomes ➡️ cells randomly pick one X chromosome to inactivate via methylation ➡️ one working X chromosome in each cell
Example of bad DNA methylation?
Fragile X syndrome: CpG island (regulatory site) is methylated ➡️ cell can’t make mRNA copy of FMR1 gene (fragile X mental retardation 1 gene) ➡️ FMRP (fragile X mental retardation protein) not made ➡️ fragile X syndrome
How do females, being mosaic, impact the severity of fragile X syndrome if they have the mutation?
Females who have most cells turn off X chromosome with fragile X mutation ➡️ most cells produce FMRP ➡️ limited impact of fragile X syndrome
Females who have most cells turn off X chromosome with working FMR1 ➡️ ⬇️ cells producing FMRP ➡️ greater impact of fragile X syndrome
What is a base analogue?
Molecules that can substitute for normal bases in nucleus acids
Usually leads to altered base pairings & structural changes ➡️ affect DNA replication & gene transcription
Which base analog is used to treat herpes virus infection?
Acycloguanosine (acyclovir)
Which base analogue is used to treat HSV & HIV?
3’-deoxy-3’-azidothymidine (AZT)
Which base analogue is used to treat leukemia?
6-mercaptopurine
Which base analogue is used to treat cancer & acne?
Which base is it an analog of? What enzyme does it irreversibly inhibit?
5-fluorouracil
analog of thymine, ➖ thymidylate synthetase
What base analogue is non-mutagenic/weakly mutagenic for eukaryotes & is mutagenic for bacteria?
What base is it an analog of?
2-aminopurine
analog of adenine
What are the 2 DNA sequencing methods discussed in class?
Maxam-Gilbert method (old, used for short fragments)
Dideoxy chain termination (uses fluorescent color tags for each of the 4 DNA bases)
What did we learn from completing the Human Genome Project?
1) 0.1% of our genome contains polymorphisms ➡️ we are all different (one gene can give rise to more than 1 protein)
2) Discovered fewer genes (25,000-35,000) than expected (100,000)
Physical structure of DNA:
? base pairs per helical turn
? diameter
? gap between bases
10 bp per helical turn
- 0 nm diameter
- 34 nm gap between bases
Physical structure of DNA: Difference between major & minor grooves?
Major groove: has more exposed space ➡️ DNA binding proteins can bind via H bonding w/exposed bases
What are the 3 types of DNA helixes?
B: Watson-crick DNA helix, R handed, main form under physiological conditions, 10.5 bases per turn
A: similar to B, R-handed, more compact, dehydrated, 11 bases per turn
Z: L-handed, zigzag structure, formed under high salt conditions, 12 bases per turn
What are intercalating agents? Examples?
Resemble ring structure of of base pairs ➡️ Insert between stacked base pairs ➡️ distorts DNA double helix ➡️ introduce mutations
Ethidium bromide, acridine orange, actinomycin D
What is plasmid DNA? What are the 3 physical structures?
Circular DNA (in bacteria), contains only a few to >100 genes ➡️ antibiotic resistance genes
Relaxed, relaxed open, supercoil
How are plasmids transferred from one bacteria to another?
Transferred via conjugation (use conjugation tube)
DNA supercoils: positive versus negative supercoil?
Positive: Add more twists to typical relaxed DNA helix (overwinding)
Negative: Remove extra twists (underwinding)
What enzyme catalyzes the formation of negative supercoiled DNA ahead of replication fork? (To relieve stress)
Topoisomerase II (DNA gyrase)
What is an inhibitor of topoisomerase?
Camptothecin
What factors can denature DNA?
pH: >11.5 and <2.3
Temperature: causes DNA to melt
Ionic strength
What ⬆️ the melting temperature (Tm) of DNA?
What ⬇️ Tm of DNA?
⬆️ GC content
⬇️ ionic strength
What is renaturation of DNA an indicator of?
What 3 things is it used to do?
Indicator of DNA sequence complexity
1) Determine frequency of certain sequences
2) Locate specific base sequences
3) Detect certain species of RNA
Where are short, highly competitive sequences of DNA found?
Telomeres (end)
Centromere (site of attachment of spindle fiber)
What are histones?
What is chromatin?
Very basic proteins with (➕) charged lysines & arginines, help to compact DNA
Chromatin = DNA + histones
What are the 5 major classes of histones?
H1, H2A, H2B, H3, H4
All exist as pairs except H1
What is a nucleosome?
Beadlike structure from resting nuclei, 10 nm diameter
Segment of DNA wrapped around histone octamer
What makes up the 30 mm chromatin filament? (Stage in condensing DNA to form chromosome)
Spring-shaped solenoid w/6 nucleosomes per turn
Stabilized via head to tail associations of H1 histone
of repetitive DNA sequences can be divided into 3 classes, based on the reassociation rate ➡️ Name & describe these 3 classes
1) Highly repetitive: 10-15% of mammalian DNA; includes tandem repeats
2) Moderately repetitive: 25-40% of mammalian DNA; includes interspersed repeats
3) Single copy (or very low copy #): 50-60% of mammalian DNA
What are the 3 subclasses of tandem repeats?
1) Satellites: Very highly repetitive, organized as large clusters (up to 100 million bp), near centrosomes & telomeres, abundant on Y chromosome
2) Minisatellites: Moderately repetitive, moderate size (9-100 bp), most highly polymorphic sequences elements yet discovered
3) Microsatellites: Moderately repetitive, short repeats (2-6 bp)
What characterizes the fragile X gene (FMR1)?
Tandem repeat sequence (CGG) near 5’ end
What is involved in a Southern blot analysis?
DNA + DNA
What is involved in a Northern blot analysis?
DNA + RNA
What is involved in a Western blot analysis?
Protein + protein
What is involved in a Southwestern (Eastern) blot analysis?
DNA + protein
5 Steps of isolation of total genomic DNA?
1) Lyse cell & nucleus w/detergent or lytic enzymes
2) Degrade DNA bound protein & nucleases w/proteinase K
3) Extract DNA w/column or phenol/chloroform
4) Precipitate DNA w/ethanol
5) Dry & redissolve DNA in aqueous buffer
What is a restriction enzyme?
Bacterial enzymes (Exonucleases and endonucleases) that cut ds DNA in sequence specific manner
Restrict entry of foreign DNA via cleaving at recognition site not found in host bacterium
Cuts asymmetrically (➡️ sticky ends) or symmetrically (blunt ends)
What does DNA ligase do?
Attaches 2 pieces of DNA covalently to each other
What does DNA polymerase do?
Uses sDNA as template, w/primer ➡️ extends DNA sequence
What does reverse transcriptase do?
Uses RNA as template, w/primer ➡️ makes cDNA
What is cloning?
What can it be used for (in lab)?
Process of making a genetically identical DNA by nonsexual means
Can make large # of DNA copies (for analysis & manipulation)
Remove DNA sequences from genome ➡️ put into vector ➡️ put into host (E. coli, yeast) ➡️ multiplied
What are examples of vectors used in DNA cloning?
Plasmid (most used)
Bacteriophage
Artificial chromosome
3 main steps of polymerase chain reaction (PCR)?
1) Denaturation
2) Annealing (forward & reverse primers)
3) Extension (DNA polymerase)
Repeat for 30-40 cycles
Detecting sequence polymorphisms example: RFLP-sickle cell anemia
What is the point mutation? What are its effects in the body?
Glu ➡️ Val, position 6 of beta-globin pro chain
Insoluble in O2-free form, crystallized, form sickle shaped RBCs ➡️ anemia, thrombotic vaso-occlusion & hemolysis
What is an example of variable numbers of tandem repeats?
Huntington’s Disease
36-125 CAG (= glu) repeats (normal 11-30) ➡️ produces huntingtin pro ➡️ “polar zipper”
What are the 2 types of replication slippage that cause micro satellite polymorphism?
1) backward slippage ➡️ insertion mutation (longer than normal)
2) forward slippage ➡️ deletion mutation (shorter than normal)
What is a DNA Library?
What are the 2 types?
Collection of cloned DNA fragments
1) genomic library: DNA fragments of entire genome of organism
2) cDNA library: only complementary DNA made from mRNA in cell
What is a DNA vaccine?
Made of single or multiple genes of immunologic proteins inserted into commercially available DNA expression plasmid
