FINAL Flashcards
is GAL4 a positive or negative TF?
positive
what happens to transcription when GAL4 is bound?
transcription is driven
yeast will use ______ preferentially (glucose or galactose?)
glucose
why is UAS significant?
where the binding domain binds
describe the DNA binding domain
recognizes specific sequence so that only those with appropriate BD are activated
what will happen if DNA binding protein is bound to BD but is cut off from AD?
transcription will not happen
bait interacts with what?
DNA binding domain
target interacts with what?
activation domain
will the bait and DNA binding domain activate transcription?
ONLY if the bait is an activation domain
will the target and activation domain activate transcription?
no, unless target happens to be BD and binds particular gene you want
what interaction must happen for transcription to be driven in yeast 2 hybrid system?
bait and target must interact and fuse *in translational frame
if the native GAL4 protein binds to UAS, what color will the colonies be?
blue
what are the two components of the assay checking for transcription activation in Y2H?
- checking for proteins that stick to one another
- checking for growth
if the AD and GAL4-BD are bound to UAS, what color will the colonies be?
blue
if you have only one fusion protein, will there be transcription?
no!
if there is a bridge between bate and target…
- interaction
- blue colonies!
the goal of a two-hybrid experiment is to….
screen a “library” for proteins that interact with a bait protein of interest
is the human library DNA or cDNA? why?
- cDNA
- why: contains only coding sequences
target protein must be in the _____ (same / different) reading-frame as activation domain and (should / should not) ____ contain non-coding sequence (5’ UTR, introns)
- same
- should not
how do we know the bait plasmid is in the cell?
the cell can now make trp
why is the NLS crucial?
transcription occurs in the nucleus (must be directed to nucleus!)
the cell that you’re starting with (for Y2H with bait and library plasmid) must be what?
trp- and leu-
why does AH109 not make his or ade?
- gene is functional but not transcribed unless there is protein-protein interaction
- transcriptionally dead (only have BD)
what selection would you perform to see if you have two plasmids in the same cell? how do you get them in the same cell?
- plate on trp- and leu-
- get them in the same cell by mating
how do you check for protein-protein interaction?
- plate on trp- leu- his- and ade- media
- if there is growth, you have protein-protein interaction!
why do you have to look for the production of both his and ade?
because the genetic rearrangement frequency is not 0
how do you determine if you have diploids?
plate on trp-leu-
what 2 main strategies were taken for sequencing the human genome?
- sequencing only expressed genes (cDNAs)
- sequencing the entire genome
why were direct clones used when sequencing the human genome?
because PCR is problematic (can make mistakes)
why was repetitive DNA hard to sequence?
- repeat sequences are homologous to each other
- undergoes a lot of recombination
- undergo recombination with each other –> anything in between is now gone/lost!
a gene can make a different protein with a different function depending on what?
how the gene is spliced
what theory explains a possible explanation as to why hundreds of genes appear to have come from bacteria?
the endosymbiont theory
_____ is the main source of mutation
sperm
what are microarrays?
a snapshot that captures the activity pattern of thousands of genes at once
T / F : a change in mRNA level means you have a change in protein level
F
T / F : knowing mRNA “expression levels” can tell you a lot about the state of the cell
T
organize these three assays in order from first to last
- Microarray
- RNA seq
- Northern blot
- Northern blot
- microarray
- RNA seq
describe the “use” of microarrays
- take mRNA from cells, put it on an array
- see where it sticks (mRNA from gene X should stick to spot X)
T / F : the intensity of color on a microarray is not related to the abundance of mRNA
F
why are spotted cDNA microarrays not done anymore?
too many variables
hybridization is driven by what two things?
sequence and abundance
if using cDNA as a probe, is the oligonucleotide the complement or the same sequence as the message?
same sequence as the message
summarize a microarray
hybridized sample –> fluorescently labelled –> stick to oligo
summarize RNA seq
- just one DNA species on a chip
- PCR on bead in chip –> bazillion copies of bead on chip –> wash bases over it –> different colors in different spots
- quantitative: based on how many copies you get, not how long the sequence is
can a microarray detect alternative splice site?
yes (technically)
do you need to know the sequence for RNA seq? for making chips?
- RNA seq: no (not necessarily)
- chips: yes
phenotype (P) = ______ + ______
genotype (G) + environment (E)
what two things can yield phenotypic variation?
mutation and selection
fine-mapping and cloning is done on a _______ basis
molecular
what is a quantitative trait? give examples
- a measurable phenotype that depends on the cumulative actions of many genes and the environment, and can vary among individuals, over a range, to produce a continuous distribution of phenotypes
- example: height, weight, blood pressure
quantitative trait loci (QTL)
the loci controlling quantitative traits
T / F : a single gene or a cluster of genes can affect a quantitative trait
T
quantitative traits are controlled by _____ genes and segregate according to _______
multiple, Mendel’s laws
T / F : QTs cannot be affected by the environment
F
allelic variations of QTs are _____ functional
fully
_______ genes control any given trait
many!
the genes involved with QTLs can be subject to
epistasis or pleiotropic effect
what is QTL mapping?
- the process of constructing linkage maps and conducting QTL analysis to identify genomic regions associated with traits
what are the objectives of QTL mapping?
- the basic objective: to detect QTL, while minimizing the occurrence of false positives
- other objectives: to identify the regions of the genome that affects the trait of interest, to analyze the effect of the QTL on the trait, determine how much of the variation for the trait is caused by a specific region, etc.
describe QTL analysis (what principle is it based on, etc.)
based on the principle of detecting an association between phenotype and the genotype of the markers
define foreign DNA
(also known as the transgene) DNA from another species, or else recombinant DNA from the samespeciesthat has been manipulated
what are transgenic organisms?
- when the transgene is present in every cell (integrated at 1-cell stage)
- an organism or cell whose genome has been altered by the introduction of one or more foreign DNA sequences from another organism
foreign DNA is also known as
the transgene
CRISPR can be used to create _____ mutants
null
transgenic animals are created by what two methods?
- DNA microinjection
- embryonic stem cells & gene knock-outs/ also Cre-loxP
if the transgene incorporates at the 2-cell stage, what will the organism be?
mosaic
what percentage of offspring from a mosaic organism will contain the transgene (if mated with a non-transgene animal)?
less than 50%
what percentage of the offspring from a transgenic organism will be transgenic (if mated with a non-transgene animal)?
more than 50%
agrobacterium is a bacterium of _______ (dicots or monocots?)
dicots
describe agrobacterium and why/how it was useful for transgenic studies
- soil-borne, gram negative bacteria
- has a naturally occurring plasmid, the Ti plasmid, that causes tumor growths on trees
describe the manipulation of the Ti plasmid
- desired gene was inserted onto Ti plasmid + of E. coli necessities to allow for growth in E. coli
- hormones controlling tumor growth were knocked out / deleted
- kanamycin marker
- select for plant with Kan resistance
- new trees will be kanamycin resistant and have no tumors
did the Ti plasmid experiment work equally well for both dicots and monocots?
no, it did not work with monocots (it does now)
drosophila have naturally occurring transposons known as
P elements
where does transposase cut?
the inverted repeats at the end of the P elements
what would happen if a transposon were to jump into a coding region? is there any control over where transposons integrate?
- create a null mutant
- no control over integration site
describe how the temporal and spatial control of transgene regulation in drosophila
spatial: tissue specific promoter
- TSP upstream of GAL4
- mate with a fly that has UAS
- transcription is driven ONLY in tissues where GAL4 is present
- works because flies do not have UAS/yeast GAL4 gene equivalent
temporal: turn on a transgene via estrogen
- tissue specific driver (GAL4)
- GAL4 VP16 complexes with HSP and cannot get into nucleus
- treat with RU486
- protein can now get into nucleus and transcription can be driven
- works because flies do not have estrogen!
describe a pluripotent cell
can become any type of cell
what must happen to get a true transgenic animal?
integration into chromosome
if transgene exists, the animal is known as a _____
founder
what is required to produce a transgenic mouse?
breakage of the mouse chromosome and ligation of transgene
how can you get more than one copy of a transgene?
transgenes can ligate to one another
is a mosaic considered a founder?
yes!
what is the most commonly used method for establishing transgenic mice?
DNA microinjection
expression will vary in transgenic offspring due to _________ and _________
position effect, copy number
describe the process of DNA microinjection
- mate mice
- harvest zygotes (about 18 hours after fertilization)
- inject DNA of interest into pro-nuclei
- zygote will have karyogamy
- implant into pseudo-pregnant mouse
CRISPR is directed by a _______
guide RNA
how can blunt end ligation be used to make targeted nulls?
if occurring in an exon, could lose “stuff” and get a frameshift mutation
describe pronuclear injection
- physically inject exogenous DNA into the pronuclei of fertilized eggs by using pulled glass needles
- mate mice
-harvest zygotes (about 18 hours after fertilization) - inject DNA of interest into pro-nuclei
- zygote will have karyogamy
- implant into pseudo-pregnant mouse
describe the transformation of drosophila
- transformation plasmid has the P element and helper plasmid has transposase
- put together, “jump” is catalyzed
- mate this white eyed fly (with the transformation) with another white eyed fly and get red-eyed offspring
the general utility of pronuclear injection has been
adding genes to mice
how did CRISPR make pronuclear injection more useful?
- CRISPR CAS9 has an RNA guide, which allows it to cut specific sequence / mutate certain genes
can you get a mutation via pronuclear injection?
yes, if the insertion is somewhere problematic
cells must do what to develop?
fuse with an intact embryo
what is the key for genetically engineering the ES cells of mice? why?
- homologous recombination
- NHR can occur anywhere
how did scientists determine if the gene targeted insertion was successful?
if it worked, the mutant should not contain the tk gene and should be resistant to neo and gan
- positive selection: treat with neomycin (selecting for recombinants)
- negative selection: treat with ganciclovir (testing for specifically homologous recombinants)
describe the placement of the engineered ES cells into an early embryo
- inject cells into the blastocoel cavity of an early embryo (ES cells are diploid, homozygous black and heterozygous for the knockout gene)
- transfer embryo into pseudo-pregnant mouse
- mouse will have homozygous white progeny and chimeric progeny
- now mate chimeric progeny with heterozygous white mouse and get some black offspring
how is temporal regulation achieved in mice? spatial?
- spatial: TSP
- temporal: the tet system
describe how the tetracycline system controls temporal regulation and why it is able to work
- if the operator is not bound, there is no expression
- must convert to positive regulatory system!
- need mutant tet repressor that binds ONLY in the presence of tet
- convert into activator
- convert from repressor to activator by switching domain (make repressor domain into an activator domain)
- so now: in presence of tet > binding > activation > transcription
- able to work because mouse does not have a DNA sequence that can bind to the regulator site in the native cell
the Cre-lox system is used for making _________ in mice
conditional deletions
describe the Cre-lox system and how it works
- have a gene of interest, make a transgene with the lox sites flanking it
- mate with mouse that makes Cre > get a transgene
- one animal has TSP and drives expression of CRE, mate with LoxP mouse > recombinase drives recombination > left with only lox!
how can there be temporal and spatial regulation of Cre?
- spatial: TSP
- temporal: sensitivity to something like Tet
- TSP driving rtTA only in specific tissues and in the presence of Tet
- deletion of genes flanked by Lox sequences are deleted spatially and temporally
tissue specific deletion is dependent on what two things?
presence of Tet and TSP driving rtTA transcription
what is genomic imprinting?
the silencing of genes through methylation
how do you know if the IGF-2 knockout worked?
resistant to G418
the pups that inherited to KO gene from mom _____ (were / were not) runts, while the pups that inherited the KO gene from dad ______ (were / were not) runts
were not, were
what can you say about mom’s gene for IGF2?
it is not transcriptionally active
was mom or dad’s gene imprinted?
moms
what are the hypotheses for mom’s gene being inactivated?
two active genes might’ve made for difficult birthing processes OR dad is making IGF-2 and mom is making IGF-2R
describe the IGF-2:H19 locus
- if IGF2 is transcribed, H19 is not, and vice versa
- insulator sequence between them > expression of one will not affect the expression of another
- silencer sequences > differential expression
- depending on germline > enhancer will act locally or on a sequence that is further away (distal gene)
- unmethylated > active gene
- methylated > gene is generally off
if A / A, what color are the mice?
brown
describe the A vy mutant
- viable yellow (other yellow mutants die)
- A vy mice tend to be obese, diabetic and are prone to cancers
- expression of phaeomelanin is developmentally regulated
how do we know the A vy mutation isn’t genetic?
1) patchiness 2) mice in the experiment were genetically identical
if the mouse is completely yellow, how methylated is IAP?
not methylated at all, allowing the promoter to be on and the mouse to be yellow (no hair cycle regulation)
describe IAP and the mouse color if there is full methylation
IAP in not on, agouti is regulated and the mouse is brown
what are IAPs? why do we think of them as transposable elements?
- defective retro-virions encoded by the members of a large family of endogenous proviral elements
- think of them as transposable elements because IAP genetic elements transposes in the genome
describe the hair cycle specific regulation (normal and abnormal)
normal
- IAP is upstream of the coding exons and is normally regulated by hair cycle specific noncoding exons
abnormal ( A vy mutant)
- IAP active: lose hair cycle regulation
describe the methylation assay for the hair cycle specific regulation
- a black animal has no locus at all (no phaeomelanin at all)
- cut up the DNA from the black mouse with BAM > don’t see 9.7 kb piece because it isn’t there, but you do see a 3.5 kb site
- cut with M (Msp1 > will cut if something is methylated or not) and H (Hpa2 > will NOT cut if something is methylated) (restriction enzymes that cut exactly the same sequence) > same bands (not methylated!)
Pseudo-agouti
- cut with M and H (H doesn’t cut, M does)
- methylation and hair cycle regulation
- see IAP band at 9.7 kb and band from the other chromosome
Yellow homozygous
- no wild-type locus
- cut with M and H (both cut)
- no methylation, no hair cycle regulation!
describe the pedigree for A vy / aa (what trend was seen)
- dad’s phenotype had little effect on distribution of pup’s phenotype
- mom did give different distribution depending on her phenotype (presumably something going on with mom’s germ cells / some in-utero effect)
was it an in-utero effect? how did they determine this?
- take fertilized eggs from yellow female and do an in-utero transfer into an aa pseudo-pregnant female
- get same distribution as before
- now know that it isn’t an in-utero effect giving the different colors > must be something in development / early embryogenesis
how can the dark patches be explained?
during early embryogenesis, that locus was methylated
if the IAP segment is hypomethylated, the mouse will be ________
yellow (no hair cycle regulation)
describe the significance of the BPA experiment on the agouti-locus mice
- wanted to see if a high BPA diet during gestation and early pregnancy effected methylation
- if it did, the BPA diet would be skewed more towards yellow (there would be more BPA fed mice in the yellow and slightly mottled groups)
- was skewed more towards yellow
describe how bisulfite was used to determine methylation percentages
- bisulfite deaminates C to produce U and does not deaminate 5meC
- if not methylated, after treating with bisulfite, C will behave like T now
- if methylated, after treating with bisulfite, C will still be C
what were the findings of the bisulfite experiment? why did they test tissue from T, B, K and L?
- methylation is a spectrum (yellow is hypomethylated, light mottled is less methylated than medium mottle, etc.)
- all three germ layers were represented in this and were similar in methylation levels, meaning that the methylation occurred very early in development and was unpredictable (not all mottling patterns are exactly the same)
did the researchers determine if the hypomethylation could be avoided? if so, how?
- BPA diet was still shifted to yellow
- BPA + methyl donor diet looked like normal diet (in terms of % A vy offspring and methylation)
