bio 305 exam 3

processes by which bacteria exchange/take up DNA

• transformation - DNA uptake from environment

• conjugation - plasmid transfer or partial genome transfer

• transduction - intake from bacteriophage

lytic cycle

1. phage attaches to host and injects DNA

2. phage chromosome replicates

3. new phage components produced and assembled

4. phage particles release by lysis from host bacteria

lysogenic cycle

1. phage attaches to host

2. phage DNA circulates and is incorporated into bacterial DNA as a prophage

3. prophage excised and lytic cycle resumes

culturing lytic phages on plates in a lab

bacteria is the “lawn” and lysed cells are “plaques”

titer

[(number of plaques)(dilution)]/(volume), with answers in p.f.u (plaque forming units)

multiplicity of infection (MOI)

average number of phage particles that infect a single bacterial cell in an experiment

low MOI (<1 phage/cell) is used to phenotype and genotype a phage

high MOI (>2 phage/cell) is used for phage crossing/recombination and complementation testing

general transduction

a phage picks up host genome on accident (same fragment length as viral genome) and transduces it to another host. . . can transfer wt allele and rescue mutant allele in host cell

cotransduction frequency depends on. . .

distance between two genes

higher frequency = closer together and vice versa

testing for complementation vs. recombination

complementation testing: one infection at high MOI

recombination testing: first infection with high MOI for recombination, second infection with low MOI to phenotype and genotype virus

seymour benzer

changed scientists understanding of genes, revealed existence of genetic fine structure, did work with lambda phage

esther lederberg

• discovered fertility factor F+, lambda phage, replica plating, and genetic mechanisms of specialized transduction

• made these discoveries but was consistently overshadowed by her husband joshua lederberg

continuous traits

on a scale, like height

variance

standard deviation, basically what the distribution of a trait looks like

Vp= Vg + Ve

quantitative traits determined by genes and environmental effects, variance in phenotype is due to variance in genotype and variance in environment

heritability

how much of a trait is determined genetically. . . phenotypic variance attributable to genetic variance

H² = Vg/Vp

if H² = 1, all due to genotypic variance, and if H² = 0, all due to phenotypic variance

midparent phenotype

average of two parent phenotypes

heritability and selection

selection doesn’t alter phenotypes of offspring if trait is not heritable

identifying QTL

looking for association between trait (which can be treated as another gene) and markers

marker not linked to QTL

no association between certain marker and trait

marker linked to QTL

association between certain marker and trait

HWE assumptions

1. infinitely large population

2. non-overlapping generations, no mixing

3. randomly mating populations

4. no mutation

5. no migration

6. no selection

genetic drift

change in allele frequencies due to chance

for alleles at low frequencies, most are found in. . .

heterozygotes

HWE for X-linked genes

males:

p = X^AY

q = X^aY

females:

p² = X^AX^A

2pq = X^AX^a

q² = X^aX^a

effects of migration in violating HWE assumptions

migration between populations with different allele frequencies will alter the allele and genotypic frequencies of both populations

assortative mating consequences in violation of HWE

in general, alters allele and genotypic frequencies

inbreeding consequences in violation of HWE

decreased frequency of heterozygotes, increased frequency of homozygotes, no overall change in allele frequency but change in genotypic frequency

haplotype

collection of mutation sequencies on a single chromosome. . . each haplotype is a unique allele

effects of mutations are. . .

situation dependent! could be beneficial, deleterious, or neutral! but always increases variation

natural selection

one of the processes that contributes to phenotypic evolution, survival of the fittest and the basis of darwin’s theory of evolution

modern synthesis

provides a theory about how evolution works at the levels of genes, phenotypes, and populations

in response to paradigm shift in biology in 1930s with morgan, fisher, dobzhansky, etc.

where does phenotype variation come from?

environmental and genetic variation, Vp = Vg + Ve

what are the two main sources of genetic variation?

recombination and mutations

fitness (w)

differential ability of individuals to survive and reproduce in a particular environment

selection coefficient (s)

1 - fitness (w)

neutralist - selectionist debate

argues over relative importance of selection and chance

random genetic drift. . .

can have large impact on allele frequencies, more than selection for low frequency alleles

founder effect

type of genetic drift where small population is established from larger population

allele frequencies in new population will differ from original population, such changes are due to chance

population bottleneck

when a relatively large population is reduced by a catastrophic event, with allele frequencies in the new population differing from the original population

founder effect is a version of this

molecular evolution

uses comparisons of DNA variability within and between species to make inferences about evolutionary relationships and past evolutionary processes

molecular clock

mutations are always happening, but the type of mutation affects the mutation rate of each protein

ex. if it’s a deleterious mutation with a strong negative effect, it will be tightly regulated and selected out of the population, creating a very slow mutation rate due to constraints

silent substitutions

aka synonymous, change in DNA sequence that doesn’t change AA sequence, usually neutral

replacement substitutions

aka non-synonymous, change in DNA that changes AA sequence and most likely subject to selection

macdonald-krietman test

developed to test for evidence of selection, only applied to protein coding regions

compares non-synonymous (dN) mutations with synonymous (dS) mutations, and if ratio of these two mutations between species is the same as within species, then there is no selection

macdonald-kreitman test results

between > within is evidence of positive selection

between < within is evidence of negative selection

selective sweeps

process by which a new advantageous mutation eliminates or reduces variation in linked neutral sites as it increases in frequency in the population

ex. teosinte and corn

experiments showing that genetic info is in DNA

• griffith’s rough and smooth bacterial experiments, found evidence for a “transformational factor” changing RII to SIII after SIII bacteria had been heat-killed and infected mice still got sick and died

• macleod, avery, and mccarty identified DNA as the transformation factor after eliminating lipids, proteins, sugars, and RNAs from extract with heat-killed SIII and RII

• hershey and chase showed that DNA is the hereditary molecule with their bacteriophage experiment, having DNA with radiolabeled P and protein coat with radiolabeled S

DNA structure

• right-handed antiparallel double stranded double helix

• major groove for binding sequence specific proteins

• minor groove

• non-specific binding proteins binding the backbone

• ribose phosphate backbone and nitrogenous bases

how long is human DNA?

almost 2 m

DNA supercoiling

• can accumulate twists and supercoils if DNA fixed at both ends, important for replication because can disrupt it

• negative supercoiling is behind the protein

• positive supercoiling is in front of the protein

essential features of genetic material

• sufficient information capacity

• ability to replicate

• ability to mutate

DNA replication direction and pattern

semi-conservative, with strands being read from 3’ to 5’ and built from 5’ to 3’

memorization hack for DNA direction

• DNA is read 3’ to 5’ like how 3 years old is the age to start reading, should be earlier than 5 years old

• DNA is built 5’ to 3’ like how 5 year olds can build better things with toys or with their imagination than 3 year olds

initiation of DNA replication

• starts at ori-initiation, AT rich area

• DNA helicase activity separates the strands and a replication bubble forms with two replication forks

what does DNA polymerase require to replicate?

DNA template, dNTPs, and 3’ OH from a primer (RNA primer from primase)

bacterial DNA polymerases

I is primer removing (exonuclease activity)

II is for DNA repair

III is for DNA synthesis

all of them proofread and can build/polymerize DNA

replisome and accessory proteins

composed of leading strand, lagging strand, DNA polymerases, DNA ligase, primase, RNA primers, DNA helicase, and topoisomerase

end replication problem

when RNA primer is removed from lagging strand, DNA gets shorter with every round of replication

telomeres

protective caps that prevent chromosome shortening, repeated DNA sequences

telomerase

• RNA-dependent DNA polymerase that elongates telomeres

• the RNA component acts as a template for telomere elongation and the protein component catalyzes the addition of nucleotides (reverse transcriptase)

• works between rounds of replication, elongating the template strand that the new lagging strand is copying from

shelterin

protein that protects the 3’ overhang because ssDNA is bad

telomerase activity

• tightly regulated

• high in early embryonic cells, germ cells, stem cells, and cancer cells

• low in somatic cells

directional selection

drives a population towards homozygosity, reduces variation within a population

effect of migration selection

homogenizes variation between populations

effect of balanced polymorphism selection

aka overdominance, selects for heterozygotes so that multiple alleles are stably present in a population, like in sickle cell anemia example

selection against heterozygotes

aka underdominance, allele frequency either goes to 1 to be a fixed allele or to 0 to become extinct. . . heterozygote does worse than either homozygote

balanced selection

selection that maintains multiple different allele frequencies within a population, occurs in cases of codominance and can be caused by overdominance or overdominance

stabilizing selection

aka purifying selection, when natural selection removes deleterious mutations and keeps phenotypes constant. . . keeps population the same

diversifying selection

aka disruptive selection, sometimes it’s advantageous to have many alleles of a gene within a population, and this type of selection keeps them around, like MHC antigen recognition protein

molecular cloning

• vector DNA (plasmid) digested with restriction enzymes, resulting in removed fragment and exposed sticky ends

• DNA of interest digested with restriction enzymes, those sticky ends combine with the vector to create a recombinant vector

• recombinant vector is plasmid with sequence of interest

benefits of recombinant vector (result of molecular cloning)

long-term maintenance, easy multiplication, easy gene expression

composition of recombinant plasmids that code for expressed protein

promoter, protein coding sequence, epitope tag that interacts with antibodies to identify and purify recombinant protein, also other helper sequences

ex. production of human insulin in E. coli

northern blot

• determines RNA length and quantity

• steps:

  • separate RNA on gel via electrophoresis

  • transfer RNA to membrane

  • hybridize with probe

what is a probe?

• short polynucleotide complementary to RNA of interest

• binds to RNA on membrane by base pairing

• typically radioactive, and radioactivity indicates presence and quantity of RNA

NOTE: CANNOT COMPARE BLOT INTENSITY ACROSS DIFFERENT PROBES

western blot

• determines protein size and quantity

• steps:

  • separate protein on gel

  • transfer to membrane

  • probe blot with antibody

• secondary antibodies label and indicate presence and quantity of protein, detected by chemiluminescence

PCR

• amplifies any DNA fragments you want

• produce DNA

• can quantify DNA or RNA (via reverse transcriptase)

• fast, cheap, in vitro

limitations of PCR

• need template DNA

• need to know sequence of flanking regions to design primers

• length limit of 10 kb

steps of PCR

1. denature DNA at high temps

2. anneal primers

3. extend primers, elongation step

cycles repeat, only products with no ssDNA overhang and length limited by primers are duplicated in subsequent cycles

typically 25-35 cycles

how do you choose Sanger sequencing primers?

sanger sequencing sequences a strand and allows inference of the complementary strand. . . to know the sequence of one strand, the primer should match the beginning of the sequence of interest, so that the strand of interest is built from the template of the complement strand

how do you make cDNA?

• ss mRNA was transcribed via reverse transcription

• product was mRNA/cDNA hybrid

• RNase degraded RNA, so that only cDNA remains, and it can be used as a template in PCR

qPCR result interpretation

• result appears as fluorescence curve

• exponential curve occurring in earlier cycles shows high DNA/RNA concentration, and vice versa showing low DNA/RNA concentration

high throughput sequencing

• sequencing on a massive scale

• millions of sequences at once

limitations of high throughput sequencing

• sequences within one sample usually anonymous

• sequences usually short (up to ~600bp)

• requires complex data analysis

genome (re)sequencing steps:

1. DNA extraction and fragmentation

2. library generation (mix of DNA fragments)

3. high throughput sequencing of each short fragment

4. aligned fragments to reference sequence - different from reference sequence means mutation at that point

genome (re)sequencing applications

• find mutations causing a phenotype

• find mutations associated with disease

• determine best targeted treatment for a specific cancer

• sequence a new interesting genome

genome (re)sequencing limitations

• very hard in the absence of a reference genome

• very hard on repetitive sequences

• difficult bioinformatics required

RNA-seq

quantification of RNA

steps:

1. RNA from tissue transcribed to cDNA

2. cDNA sequenced and mapped to genome

3. many matching fragments show high RNA concentration and sometimes high expression (NOT ALWAYS), and vice versa for few matching fragments

advantages of RNA-seq

• know RNA accumulation from ALL genes

• analyze transcription and RNA processing

limitations of RNA-seq

• need genome sequence

• less abundant RNAs hard to study

• often overinterpreted as a measure of gene expression, doesn’t have to mean this

ChIP-seq

shows protein-DNA interactions

steps:

1. cells lysed and bound with antibodies

2. DNA from these cells sequenced and mapped to genome

3. many matching protein to DNA regions means protein was bound and vice versa

advantages of ChIP-seq

• find all binding sites of a protein

• can study posttranslational protein modifications

limitations of chIP-seq

• need an antibody specific towards protein

• need a good negative control

• limited resolution