gene expression Flashcards
gene expression
all cells in body have same dna but very different morphology
cell differentiation
specialization of cells where they commit to specific morphology
what kinds of cells do stem cells differentiate into
ectoderm, mesoderm, endoderm
hematopoetic stem cells
stem cells of blood cells
totipotent
can differentiate into any type of cell
- applies only to zygote through stage of morula
pleuripotent
differentiate into any germ layer (ecto,endo,meso) obtained from blastocyst
multipotent
describes adult stem cells, can differentiate into several types of a specific cell
olgiopotent
can only differentiate into a few types
apopstosis importance to development
helps establish boundries between organs and structures
operon
gene expression in prokaryotes
- can be under positive or negative control
negative control operon
repressor prevents transcription by binding to an operator
operator
sequence upstream of the first protein coding region
positive control
activator stimulates trascription
lac operon
negative control and inducible
gives ecoli the ability to metabolize lactose
when lactose is present and glucose is active, lactose is metabolized
promoter region
upstream of operator
what happens to lac operon when lactose is NOT present
the represor is bound to the operator and prevents transcription of lactose metabolizing enzymes
when lactose is present what molecule binds to the repressor?
allolactose , which frees the oeprator and transcription may begin
why is the lac operon typically turned off?
because metabolizing lactose when glucose is available is energetically unfavorable
CAP binding sequence
bound by high levels of cAMP
positive control, when bound lactose metabolism is enhanced
what is the function of cap binding proteins
they promote protein synthesis
what is derived from endoderm
internal lining of respiratory tract, digestive tract
liver, pancreas, lungs, epithelial linings
what is derived from ectoderm
epidermis, brain, spinal cord,
what is derived from mesoderm
dermis, heart, bone, bonemarrow, muscle, urogenital system
what is the outcome when no lactose is present and glucose is present?
the repressor is bound to the lac operon and lactose is not metabolized
what happens when lactose is present and glucose is not
cap binds to cap binding sequence, and repressor is bound to allolactose so lactose can be metabolized
trp operon
contains genes that synthesize tryp, is turned off when trp is part of the diet
- trp binds to repressor which binds to the operator and prevents tryptophan synthesis
which operon is an example of repressible negative
trp, turned off in the presence of tryptophan which is the envirnmental change and is naturally on
promoters
upstream regions of dna that initiate transcription
bind to proteins that help recruit RNA polymerase to initiate transcription
example of common eukaryotic promoter
TATA box and CAAT
TATA binding protein
binds to tata box and contributes to the binding of RNA polymerase
transcription factors
proteins that regulate expression by binding to a specific sequence of DNA that recruit proteins that play a role in acetylation and methylation
enhancers
allow increased gene expression , not necessarily close upstream of coding region , bring transcription factors closer to gene
silencers
opposite of enhancers, where repressors bind
histones
positively charged proteins bound to dna
heterochromatin
tight structure of histones and dna
euchromatin
lose binding of dna and histones (beads on a string)
- associated with higher levels of transcription
histone acetyltransferases
modify histone structure by transfering an acetyl group to histones and making them less positive to loosen binding to dna via LYSINE residues
what amino acid residue of histones are acetylated
lysine
histone deacetylases
remove acetyl groups from histones, making them more positively charged and repressing transcription
dna methylation
methyl group is added to the dna via cystine or adenine and decreases transcription
- related to epigenetic changes
non-coding rna
rna that is not translated into protein like trna or introns
- can be involved in gene expression
major non coding rna
small interfering rna and mircorna
siRNA structure
short double stranded rna
microRNA
single stranded and long basic hairpin loop
importance of miRNA and siRNA
they can silence genes by interrupting expression between transcription and translation
cancer
abnormal gene expression
tumor
proliferation of cells
benign tumor
localize
maligant tumor
metastasizes
tumor initiation
changes that allow a single cell to proliferate abnormally
- develop ability to bypass cellular checkpoints
tumor progression
cell devlops ability to proliferate more aggressivley and malignant cells mutate
tumor promoters
induce the growth and proliferation of cells for growth and division
oncogenes
promote abnormal growth
tumor viruses
usually contain retroviral oncogenes that stimulate cell proliferation
(HPV)
protooncogenes
genes that act as oncogenes after a mutation
tumor supressor genes
inhibit oncogenesis (p53)
restriction endonulceases
cleave dna at recognition sites that are palindromic that leave sticky ends after cleaving
palidrome
has symmetry and can be cut down the middle
the 5’ to 3’ are the same on each half
recombination
use an endonuclease to cleave dna and combine two different genomes that are sealed with dna ligase
vectors
dna molecules that carry info into a cell that can replicate it
types are plasmids and bacteriophages
types of vectors
plasmids (bacteria) bacteriophages (virus)
plasmids
short circular dna molecules that replicate INDEPENDENTLY from bacterial genome and can confer resistance. also can be recombinant
antibiotic resistnace genes
typically added to plasmids so researchers know which bacteria took plasmid
reporter gene
codes for a product leading to an obvious phenotypic change that contains recognition sites for restriction enzymes
- so only bacteria that are not recombinant will posess reporter gene, and bacteria that is recombinant will not
restriction site
recognition site for a restriction enzyme to cleave dna
antibiotic resistance gene
allows selection of bacteria that have taken up plasmid
reporter gene function
distinguishes bacteria with recombinant plasmid from those with nonrecombinant plasmids
disadvantage of using plasmids for cloning dna sequences
only 2-4 kB of dna
bacteriophage as vectors
sequence genomes as large as 15 kB
major difference between bacteriophage vectors and plasmids
bacteriophages strip non-essential genes to carry recombinant sequences
cDNA
complimentary dna, cloning rna
first synthesize a dna copy of the rna using reverse transcriptase which is cdna and is then ligated to dna vector
major difference between cnda and dna
dna has coding and noncoding regions, cdna is from an oringial mrna trascript and has coding information only
transgenic
genome modified
gene therapy
splice in a functional copy of gene
stem cell therapy
embryonic stem cells used to regenerate tissue
gel electrophoresis
analyze nucleic acids by SIZE
- suspend charged molecules in agorose and apply electric field with positive and negative ends
- will move toward positive end and speed depends on size
DTT
agent that degrades disulifde bridges on agarose
hybridization
ability of single stranded dna to form base pairs with complimentary sequences
- dna annealed at high temps
- used in PCR
hybridization probe
identify target sequences by using specific dna or rna sequence that is radioactive and anneal the radioactive strand to complimentary sequence
blotting technique mnuemonic
SNOW
DROP
blotting technique
molecule of interest undergoes gel electrophoresis to seperate by SIZE
what macromolecule is examined with western blot
antibodies
DNA microarrays
large scale version of southern blot
- global analysis of dna or rna
glass slide with cdna and more than 10000 dna sequences can be shown
sanger method of dna sequencing
premature termination of dna synthesis resulting from inclusion of chain terminating dideoxynucleotides with no 3’ OH group
- dideoxynucleotides radioactively labeled and ran through gel electrophoresis to seperate
PCR
amplification of a dna sequence in vitro
- dna doubles each round
pcr primer
initate dna synthesis at desired point
- synthesized oligonucleotides
- 2 primers in opposite directions
annealing of pcr
template dna is heated above 95C to seperate dna then themp os lowered to allow primers to pair with compliment dna , then dna ploymerase uses the primers to make new strands
dna polymerase in pcr is
a heat-stable enzyme that can operate above 75C
- taq polymerase
can cdna be used for pcr
yes, just need reverse transcrptase step prior to pcr to make the cdna
