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CH 18, 19, 20 (ch 18: regulating gene expression (regulation of…
CH 18, 19, 20
ch 18: regulating gene expression
regulation of prokaryotic genes
regulate enzyme activity: affect protein production (feedback inhibition)
regulate enzyme production: turn genes on/off (transcription)
operon
- operator, promoter, genes
use one control for multiple genes
operator
- switch that turns promoter on/off
repressor
- protein that blocks transcription when binded
corepressor
- bind to repressor to switch off
regulatory protein
- produces repressor, has own promoter
negative gene regulation:
repressible
(trp operon) - usually operon on, repressor not bound, trp produced
trp present: bind and activate repressor --> operon off, trp not produced
inducible
(lac operon w/ lactose) - usually operon off, repressor bound and active, no lac produced
lac present: bind to repressor --> repressor inactive, operon on --> lac produced
positive gene regulation
lac operon (w/ glucose & cAMP)
low glucose, high cAMP: cAMP bind and activate CAP, more transcription
high glucose, low cAMP: CAP inactive = little transcription
regulation of eukaryotic genes
regulating chromatin structure
histone modification
- add -COCH3 to histone tails --> tails don't bind to nucleosomes --> unpacks/loosen chromatin
add methyl (-CH3): DNA packs
add phosphate to methyl: DNA unpacks
DNA methylation
- add methyl to DNA bases --> long term inactivation
regulation of transcription
control elements
- noncoding DNA, where transcription factors bind
role of transcription factors:
proximal
- control elements close to promoter
distal
- control elements very far from promoter
enhancers
- distal control elements bend DNA to control promoter
regulation of post transcription
alternative RNA splicing
- diff mRNA from same primary transcript b/c take out diff introns
proteasomes
- recognize ubiquitin and degrade proteins
ch 20: biotechnology
studying the sequence, expression, and function of a gene
determining gene function
single nucleotide polymorphism (SNP)
- variation in base-pair in at least 1% of population
in vitro mutagenesis
- mutate growing genes in test tube
RNA interference (RNAi)
- build RNA that damages mRNA
analyzing gene expression
groups of genes:
DNA microassay
- look at difference b/w normal and mutant cells
put cDNA in biochip and use florescent to detect
studying single genes:
RT-PCR
- use mRNA --> cDNA, then use PCR to multiply
quicker and use less mRNA than Northern blotting
in situ hybridization
- track down mRNA with probes in original organism
gel electrophoresis & southern blotting
Southern blotting
- identify specific DNA sequence
gel electrophoresis
nitrocellulose paper blot to transfer DNA
paper in bag --> put radioactive probe that binds to target sequence
Northern blotting
- same process but w/ mRNA
RFLPs
- diff DNA sequence at the same allele; causes diff fragment length
identified with Southern blotting; used as genetic markers
gel electrophoresis
- separate nucleic acids and proteins based on size
cut up DNA and add to cathode side
DNA released and go towards anode - smaller DNA mover faster/farther; result: banding pattern w/ diff lengths
look at actual DNA w/ florescent
DNA cloning to copy a gene/DNA segments
cloning a eukaryotic gene in plasmid:
cloning vector
- DNA replaced with foreign DNA then replicate in host cell
bacteria plamids good choice b/c easy to obtain, insert DNA, and put back in bacteria; multiply very quickly
nucleic acid hybridization
- base pairing gene and complementary DNA sequence
nucleic acid probe
- ssDNA or RNA that base pair bonds to target sequence
cells from well --> nylon membrane that seperates DNA strands
radioactive probes bond to ssDNA on membrane
radioactive DNA wells detected --> find clone with gene of interest
obtain gene of interest and plasmid with restriction site
cut both DNA w/ same restriction enzyme
mix fragments together --> some recombinant some not
use mutated bacteria to identify recombinant plasmids
making recombinant DNA:
restriction enzymes
- cut up foreign DNA
restriction site
- specific DNA
sequence for each enzyme
restriction fragments
- pieces of
DNA that are cut out
sticky end
- fragments with staggered end; binds with comp sticky end from diff DNA
DNA ligase
- binds covalent bonds in sugar phosphate backbone
gene cloning
- producing multiple copies of one gene
purposes:
amplify specific genes
produce more specific protein