week 10 (gene exp. in euk. + recombinant dna) Flashcards
name: mechanisms of gene regulation in eukaryotes
IN NUCLEUS BEFORE TRANSCRIPTION
- regulatory prot.
- regulatory sequences (enhancers and silencers)
- chromatin struc
- methylation
- alternative promotors
IN NUCLEUS AFTER TRANSCRIPTION
- 3’ polyadenylation
- splicing
- 5’ capping
AFTER SPLICING
- small RNAs (introns and exons) that influence mRNA stability
- factors that influence mRNA transport and stability
IN CYTOPLASM
- posttranslational modifications
- binding of regulatory molecules
- regulation of protein stability
explain: enhancers and silencers
- far from regulatory seq.
- can be upstream or downstream
- bind regulatory prot. and interact w/ proteins bound to promotors
explain: sonic hedgehog gene
- discovered in drosophila
- mammals have 3 hedgehog homologues
⤷ one = sonic hedgehog (SHH) - example of enhancers
- directs dev. of limbs and brain
⤷ 2 enhancers (one for limbs, one for brain) - regulation of SHH depends on what transcription factors are expressed
explain: GAL4-UAS system
- in yeast
- transcription of genes in galactose metabolism
- regulated by enhancers
- happens when galactose is the only sugar available
⤷ break down galactose
explain: structure of GAL4-UAS system
- genes: GAL2 -> 1 -> 10 -> 7
- each GAL gene has its own promotor and enhancer
- GAL4 = always present in cells + activates transcription by binding of UAS elements
**GAL4 = inactivated when bound to GAL 80
name: order of galactose breakdown from GAL4-UAS system (which are prod. by which gene in the system?)
outside galactose
permease (GAL2) ->
inside galactose
galactokinase (GAL 1) ->
UDP galactose
UDP galactose-4-epimerase (GAL10) ->
UDP glucose
galactose 1-phosphate uridyltransferase (GAL7)
glucose-1-phosphate -> glycolysis
explain: GAL4-UAS system when galactose absent vs present
ABSENT
- GAL4 bound by GAL80
- no transcription
PRESENT
- GAL3 binds to GAL80 -> releases from GAL4
- activates transcription
explain: insulator seq.
- located between enhancers and promotors of genes
- block enhancer activity towards a specific promotor
⤷ redirect it to another gene
explain: insulator effect on chromosome 11 of humans
- on maternal chromo.
⤷ insulator blocks IGF2 expression
⤷ directs enhancer to H19 instead - on paternal chromo.
⤷ methylation inactivates insulator + blocks H19
⤷ enhancer driven to IGF2
question: (RNA interference) microRNA vs short interfering RNA?
- double stranded RNA gets processed by enz. (Dicer) -> microRNA
- micro = made from double stranded RNA expressed w/in a cell
- short interfering = made from ds RNA expressed from external sources
explain: RISC
- RNA induced silencing complex
- fragments of RNA (micro or short interfering RNA) bind to it
- one strand gets discarded, the other -> guide strand
- 3 mechanisms for silencing
⤷ destroy the mRNA
⤷ block transcription of mRNA (eventually also degrades mRNA)
⤷ direct enz. to nucleus to silence transcription
explain: mechanisms of RISH for gene-silencing (3)
- RISC guide RNA strand binds to mRNA
- destroys mRNA
- binds mRNA and prevents translation
- RISC directs chromatin modifying enz. to nucleus where transcription of gene happens
**mRNA gets degraded in 1 and 2
explain: Dnase 1 hypersensitive sites
- regions sensitive to Dnase 1 digestion
- have euchromatin
⤷ looser -> more sensitive - transcriptionally active
- method of detecting transcriptionally active areas
⤷ see the digestion = more euchromatic
explain: chromatin immunoprecipitation
- method to isolate DNA bound by prot. of interest
- helps ID where transcription factors bind and regulate gene expression
- formaldehyde forces prot. to bind to DNA
- treat w/ antibodies -> antibodies bind to prot. of interest
- prot. precipitates and can be washed off
- left w/ DNA
question: how does consuming royal jelly result in a queen bee?
- eating more jelly -> reduces methylation -> activates queen genes
- epigenetic changes
⤷ queen and workers can be genotypically identical
question: how does gene expression influence circadian clock?
- genes period (per), timeless (tim) get transcriptionally activated by genes clock (clk) and cycle (cyc)
- per and tim highest at night = inhibit clk and cyc
- cryptochrome = blue light receptor
⤷ inhibits tim when there’s light -> allows clk and cyc to activate per and tim - makes cycle synchronized by light
explain: restriction enz. for recombinant dna
- nuclease enz. that recognize specific DNA seq. and cut DNA
- some make cuts that cause sticky ends (overhang)
⤷ frag. w/ sticky ends can combine
question: what’s the number of nucleotides between cuts for a restriction enz.?
4^n where n = number of nucleotides in recog. sequence
explain: restriction mapping with restriction enzymes
- exposed DNA to various restriction enz and analyzed frag. size using gel electrophoresis
question: if a circular plasmid was digested by a restriction enz. w/ 3 cut sites, how many fragments would there be?
3
**circular: number cuts = number fragments
linear: number cuts + 1 = number fragments
explain: molecular cloning
- isolated fragments can be inserted into a vector (plasmid) and introduced into a biological system to amplify the DNA
- copies of the DNA frag. = clones
- combine plasmid (vector) and DNA frag. w/ DNA ligase to make a recombinant DNA clone
- insert plasmid into biological system
- allow recomb. plasmid to replicate
explain: plasmid insertion process into e.coli (plasmid into biological system)
- usually by the process of transformation
- generate competent cells + keep them on ice
- expose them the heat shock
⤷ helps plasmid enter bc it widens pores of competent cells - return to ice
⤷ closes pores to prevent leakage
explain: verifying insertion of DNA in plasmid w/ blue-white screening
- some plasmids have lacZ gene w/ restriction cut sites
- if plasmid successful -> lacZ will get frameshift mutation causing gene inactivation
⤷ look for white colonies to show plasmid has inserted - plate cells w/ X-gal
⤷ when X-gal is broken down -> produces blue dye - blue colonies = insert frag. is missing in plasmid
- white colonies = insert is present
explain: verifying insertion of DNA in plasmid w/ size
- grow a bunch of colonies separately
- extract plasmid DNA
- digest plasmid DNA w/ restriction enz
⤷ inspect the frag. sizes - usually: recomb. frag. = larger than non recomb.
**method has more trial and error but works for more general vectors
