quiz #1 Flashcards
Reasons for Mendel’s success
- most people at the time studied complex traits –> complex results
- he studied simple individual traits
Mendel conclussions
- interpreted his quantitavely based on probability
- alleles of unlinked genes sort independently (genes on teh same chromosome often exhibit various degrees of linkage depending on how close they are together –> chromosome mapping)
Advantages of peas for mendel’s work
- many variants available from commercial sources
- normally self-fertilizing (true breeding lines like homozygous)
- easy to cross
- crosses not easily contaminated with other pollen
- relatively fast life cycle
simple dominant vs recessive alleles
dominant- often produce a functional gene product
recessive- often do NOT produce funcitonal gene product because most recessive genes are missing something (this is why most disease genes are recessive –> from mutations that eliminate gene function)
haplosufficient
one copy is enough for normal function in heterozygotes
types of loss of function mutations
null/amorphic: no functional gene product
leaky/hypomorphic: small amount of wt product or function
conditional: only manifest under a particular condition
**dominant negative **
dominant negative mutation
function of protein complex is altered by mutant gene product that interacts abnormally with its usual partners –> malformed protein complex
types of gain of function mutations
hypermorphic: more activity per allele than usual; often due to increase in gene copy number, higher transcription/allele or loss of inhibitors
neomorphic: mutants acquire novel gene activities that are not found in wild type; usually dominant
incomplete dominance
full contributions from both parents are required for full phenotypic expression
one copy works, but 2 copies work better
co-dominant expression
each allele produces a unique product which does not mask expression of the other
all alleles identified directly from DNA sequence data are always co-dominant (also applies to simple sequence repeats)
pleiotropy
the production, by one particular mutant gene, of unrelated multiple effects at the phenotypic level
multigenic trait
more than 1 gene specifying a given phenotype
positive genetic interaction example
suppression:
one allele is a suppressor for another mutant allele. Second mutation effectively “reverses” the effect of a mutation of another gene –> WT
negative interaction example
synthtic lethality
synergistic phenotype= contribution of 2 or more genes to a ……. exceeds the expectations from sum of their individual effects
WT: fully binding; fully functional
Mutant A: partial binding; functional
Mutant B: partial binding; functional
double mutant: binding impossible; nonfunctional
what does synthetic lethality suggest about molecular mechanisms?
share a common function (if cut off both, youre not going anywhere)
when does recombination happen?
prophase 1
why is recombination needed?
allows unfavorable alleles to be eliminated and favorable alleles to accumulate
forward vs functional genetics
forward: phenotye –> genotype
reverse: genotype –? phenotype
model organisms considerations
relevance and tractability
genetic and biochemical perspective
commonly used model organisms
e coli, budding yeast, round worm, fruit fly
zebra fish, mouse, thale cress plant
how are polynucleotide chains conneted in DNA and RNA?
phosphodiester bonds
DNA’s relation to:
2’ -OH
3’ -OH
2’ : DNA lacks
3’: where things are added on to
why use DNA for long-term storage of genetic info?
because DNA is not rapidly hydrolyzed under basic conditions, unlike
RNA because of the 2’ -OH
RNA sense vs antisense
RNA: top strand, same as sequence message
antisense: bottom/coding stand, complementary to message
often only top/sense strand is written
chargaff rules
suggested base pairing and replication mechanism
A=T, G=C
B-DNA
- right-hand double helix
- 20 A wide
- 3.4 A vertical rise/ bp
- 10.5 bp/turn
what is DNA stabilized by?
hydrophobicity, H-bonds, base stacking
why does DNA have T and not U?
C deaminates to U
U is removed from DNA by uracil-DNA glycosylase
why is 5’ methyl C often a hot spot for mutation?
5’ methyl C deamintes to T
recognition element
a-helix protein can easily bind to major grove IFFFF it has the correct pattern of H-bonding
R-group amino acids that can also make hydrogen bonds with base edges
- polar and hydrophilic R-groups
- Asn, Gln, Glu, Lys, Arg
how are polynucleotide chains flexible?
rotation around glycosidic bond
sugar pucker
- pentose pops up; large impact on chain geometry
- C-2’ endo predominant in DNA
- C-3’ endo predoimant in RNA
propeller twist
- allows bases to stack in a way that excludes more water
- constrained by H-bonds
approx 3.2 for A=T
6.7 for G=- C
exact compromise depends on local sequence
GC rich regions vs AT rich regions
naturally “bent” DNA
GC rich: have WIDER minor groove (GC greater twist than AT)
AT rich: have NARROWER minor groove
sharp kinks at boundary of regions with greater propeller twist
eliminates need for certain regulatory proteins
TATA box bends DNA at 90 but if DNA is already bent, dont need TATA box
A-DNA
- short, fat, tilted, different pucker
- predominates at low moisture
- has low water content
- naturally found in RNA/DNA and RNA/RNA helices (because RNA does 3’ pucker)
how do extremophiles survive of 80 C and pH3?
by adopting the complete DNA in the A-form and therby aids protein to encapsulate DNA
when does Z-DNA form?
- at high [salt]: poly d(GC); poly d(AC)+poly d(GT)
- under physiological conditions: poly d(GC) mainly form B-DNA
- methylation of carbon 5 of G (m5G): shifts equilibrium to favor Z-DNA by binding a hydrophobic patch
- underwinding caused by polymerases and helicases
predicted effect of B–> Z shift?
radical change in gene expression
what happens if you put methyl hydrophobic pocket?
can more easily transition into Z-DNA
which DNA occur in nature?
All 3: B, A, Z
hairpins and cruciforms
- SS DNA can readily form hairpins but cruciforms usually not favored under phisiological conditions
- SS binding proteins prevent hairpins during DNA replication
triplexes vs quadraplexes
trip: “Hoogsteen” pairing; 3-stranded proteins
quad: 4 GC rich strands; form on telomeres with assistance of proteins
how does RNA fold?
- very compact –> less exposure to water (good)
- structures can be predicted by having the most legative deltaG
- greater structural flexibility of RN allows G=U (wobble) base pairs
pseudoknots
- make different sets of proteins byshifting reading frames in RNA
- cause ribosomal frame shifting in HIV, to allow production of the reverse transcriptase needed for viral replication
RNA features that increase secondary structure stability
- unique recognition sites for aminoacyl-tRNA synthetases ribosomal proteins
- A-form: closer phosphates
- 2’ -OH hydrogen bonds
- coordination with metals
- direct to oxygen on an adjacent ribose
- via a water, between a 2’ OH and a phosphate oxygen
RNA tertiary structures
- vast array, bind ligands and catalyze chemical reactions
- self-splicing introns
- riboswitches (can act different based on of its bound to a drug)
- form peptide bond in robosomes (done by RNA chemistry, not any proteins)
DNA hybrid formation
DS DNA melted and re-annealed
higher Tm: higher UV absorbance and lower viscosity
increased absorption = hyperchromic effect
Tm depend on?
- GC content
- [salt] (if low, repels)
- pH (low= depurination- purines bases fall) (high= disrupts H-bonds)
- chaotropic agents
- for short sequences, length is importnt
high salt= phosphate shielded
low salt = phosphates not shielded –> ripped apart
between dsRNA and dsDNA, which is more stable?
dsRNA
what nucleic acids can form hybrids?
from different species (alive or extinct)
southern vs northern blotting
southern: separate DNA with restriction enzyme on native non-denaturing agarose gel (look at complex structure)
northern: separate RNA on denaturing gel (to have single strands so migration is based on size and not structure)
DNA or RNA on solid supports must be denatured before hybridization with labeled probe
restriction enzume to cut at wide ranges of sizes to see better in gel
stringency of hybridization
- conditions used during a nucleic acid hybridization experiment that determines how closely a probe sequence must match the target sequence to bind
- how close are you to Tm? how much salt youre gonna put and at what temp?
DNA supercoiling
- when ends are fixed like in circular bacterial chromosome or the loop domains of eukaryotic chromosomes
- underwinding: negative supercoiling –> facilitates strand separation (predominant in DNA)
- overwinding: positive supercoiling
linkage number
linkage= writhing # + twisting #
= number of times DNA strands twist about each other (a fixed number)
supercoiling alterations
- topoisomerases alleviate the stress of replication and transcription by introducing or relaxing supercoils –> allows DNA to maintain an underwound state
- palindromic sequences allow cruciform DNA
Type 1 topoisomerase
- break 1 strand of DNA, pass unbroken strand through, and religate broken ends
- changes linking number by 1 (delta Lk=1)
- reaction cycle involves formation of an enzyme bridge that prevents uncontrolled relaxation of DNA
- does NOT require ATP
Type 2 topoisomerase
- break both strands of DNA, pass unbroken strand through, and religate broken ends
- changes linking number by 2 (deltaLk=2)
- requires ATP
bacterial DNA gyrase
introduces negative supercoils
Eukaryotic type 2 topoisomerase
- do not introduce - supercoils but can relax + and - supercoils and untangle DNA by allowing one strand of DNA to pass through another
how do topoisomerases increase/decrease underwinding?
by changing linking number (Lk)
topoisomerase inhibitor
Ciprofloxacin- for bacterial infections including Anthrax. Blocks DNA passage
Topotecan- antitumor agent, block human topo1
electrophoretic mobility of linear DS DNA is determined by …………..
length
because charge of nucleic acids comes from phosphodiester backbone
1% vs 2% gel
1%: if small, go right through and stack at the bottom
2%: avg pore size is smaller, so small stuff will get stuck where supposed to
why cant you go higher than 0.5% gel?
because at some point, friction does not make that much difference
standard DNA gel electrophoresis vs pulse-field electrophoresis
standard: can resolve fragments up to 50kb using 0.5% gels, which are very soft
pulse-field: separates DNA up to 10 Mb, using 1% ges, which are much easier to work with
- DNA slowly zig-zags down the gell
- every time the current shifts direction, DNA must re-orient to align with field before it can migrate
- small DNA reorients more quickly and thus moves faster
what is used to visualize DNA with UV light?
- ethidium bromide (EtBr): detection limit 0.5 to 5.0 ng/band; toxic
- GelRed: less toxic because unlike EtBr, does NOT cross cell membranes
both intercalate between the basepairs of DNA
blot hybridization
- detect specific DNA and RNA sequences
- charge on nucleic acids allows them to bind + charged surface (like nitrocellulose)
housekeeping genes
- used as controls on Northern blots
- PECAM-1 (mRNA)
- GAPDH (mRNA)
recombinant DNA technology
- get DNA segment to be cloned (restriction enzyme and size selection after electrophoresis; direct synthesis)
- select DNA vector that can self-replicate (usually plasmid with antibiotic resistance gene)
- join 2 DNA fragments covalently (DNA ligase, Gibson assembly)
- transform recombinant DNA into a host (typically E. coli)
- select hosts that have recombinant DNA
restriction enzymes
- have different recognition sites and cut DNA differently
- chop up foreign DNA if it comes with the wrong pattern
what does cleavage of palindromic sequence generate?
DNA with complementary ends
cloning vectors key features
origin of replication (high vs low copy #)
selectable marker (antibiotic resistance)
insertion site for foreign DNA (polylinker)
Gibson assembly
glues things together without need for compatible (sticky) ends in a single isothermal reaction
1. exonuclease chews 5’ to 3’
2. single strand regions anneal
3. gaps filled by pol 1 and ligase
DNA libraries
large collections of recombinant DNAs, each with same vector but different inserts
2 types
1. genomic library: entire genome is represented
2. CDNA library: expressed RNAs from particular cell or tissue-type are represented
what does reverse transcriptase (RT) do?
generates complementary DNA (cDNA) from RNA template
Hairpin primed 2nd strand cDNA synthesis
- reverse transcriptase form loop that can prime 2nd strand synthesis
- forms due to endogenous RNase H actiivty of AMV reverse trasncriptase
- simple, but unpredictable because the 2nd strand priming event can occur randomly along the mRNA template
- must cleave loop with S1 nuclease
template switching RT
when RT reaches the end of mRNA it often adds a few Cs (non-templated)
these can bind to G residues of a Template Switching (TS) oligo
RT can then extend across the TS oligo giving common sequence on the 3’ of all the transcripts
PCR elements
DNA template, primers complementary to ends of target, dNTP, thermostable DNA polymeaser (pol)
PCR steps
- assemble reaction mix minues pol on ice
- add polymerase and start first melt cycle
- anneal at temp that only allows primers to bind correct sequence (usually 5C below primer Tm)
- elongate (72 C for 1 min/kb)
- repeat (heat, anneal, elongate) 30-35x
- long final elongation to finish all ends
heat-stable DNA polymerase
Taq!!!!!: can remain active after every heating up step; does NOT have proofreading activity and thus makes mistakes
Physion: have proofreding activity and 50x lower error rate
what does CODIS show?
highly polymorphic regions in chromosomes
