Exam 3 pt 2 Flashcards
insulin is made with bacteria process
- The two different polypeptides (A chain and B chain) are inserted into different plasmids and then put into bacteria (transformed).
- The cells are cultured. Purified B-galactosidase-insulin fusion proteins.
- Treat with CNBr. CNBr cleaves the peptide bond after methionine.
- purify A and B chains.
- Refolding and disulfide bond formation
the cycle threshold is reached when
the accumulation of fluorsecense is much greater than the background fluorescence detected by a thermocycler
Why do many researchers include GADPH in their Northern Blot experiment?
Because GAPDH is expressed at the same level in all cell types, researchers include it in their Northern blot experiments as a control. It confirms that the same amount of RNA is present for each cell type and that the Northern blot was successful.
Why are The A and B chains are linked to 𝛽-galactosidase in production of insulin
This is done so that the fusion protein is stabalized. If the chains were not linked to 𝛽-galactosidase, then they would be degraded, and 𝛽-galactosidase increases stability.
why are The purified proteins are treated with cyanogen bromide in insulin making
This is done to cleave off the beta-galactosidase portion of the protein. This cleaves the peptide bond connecting the fusion proteins to separate them into separate proteins. This allows the protein to form into insulin and fold properly.
biological control
the use of living organisms or their products to alleviate plant diseases or damage from environmental conditions.
* Ex: Bt is natural occurring and kills caterpillars and insects but is nonharmful to humans and plants. Put on crops
bioremidation
the use of microorganisms to reduce enviromental pollutants
Ex: microorganisms to treat and degrade sewage
biotransformation
enzymes produced by the microorganism transform the structure of the toxic pollutant
how is a gene knockin made in mice
- gene of interest is cloned and flacked with peices of DNA that are homologous to a noncritical site in the mouse genome
- cloned DNA introduced into a mouse oocyte
- gene of interst inserts into the noncritcal site by homologous recombination
gene reduncy might mean
that a gene knockout has no effect
procedure of making a protein be screted in milk
- insert gene in plasmid next to milk specific promoter
- DNA injected into sheep oocyte
- female transgenic offspring secrete protein in milk
- purify protein from milk
cloning of animal how
- donor sheep’s mammary cell is extracted and grown in flask
- another sheeps unfertilized egg is extracted and nucleus removed
- mammary cell and egg with no nucleus are fused with electrical pulses
- egg starts turning into an embryo with donor DNA and egg proteins
- embryo put in surrogate
- identical sheep born
dolly age problems
seemed to be as old as donor bc shorteing of telomeres matched the donor
stem cells characteristics
- capacity to divide
- differentiate into anything
totipotent cells
can give rise to all cell types
pluripotentent
can turn into most cell types
multipotent
several cell types can turn into
unipotent
can only turn into one cell type
hematopoietic stem cells are
multipotent
most ES cells from
IVF
most EG from
aborted fetuses
induced pluripotent stem cells
made pluripotent, but how to make them differentiate?
DNA microarray is
plastic slide with many sequences of DNA
microarray can
identify genes that are transcribed
DNA fragments on a microarray can be
- amplified by PCR and then spotten onto the microarray
- synthesized directly on the microarray itself
how is microarray used to study gene expression
- islate mRNA
- make cDNA that is fluorescently labeled
- hybridize cDNA to microarray
- view on a lser scanner
*
microarrays make it possible
to study the expression of the whole genome under different enviromental conditions
goal of ChIP
to determine where target protein binds in genome
ChIP steps
- add formaldehyde to crosslink proteins to DNA
- lyse the cells
- sonicate DNA into small peices
- add antibodies that recognize the protein of interest
- the antibodies are bound to heacy beads
- centrifuge the sample
- pellet contains protein of interest, linked DNA
- uncrosslink the pellet
two options:
* if have an idea of DNA: conduct PCR of DNA
* if PCR amplifies then the protein of interest was bound to the DNA region that is flanked by the primers
OR
- attach linkers
- conduct PCR with linkers and flurescence
- do microarray
RNA seq used to
study the simultaneous transcription of many genes
transcriptosomes
the set of all RNA molecules that are transcribed in one cell or a population of cells
RNA Seq technique
- isolate RNA
- break RNA into small samples
- add linkers to the RNA
- synthesize cDNA with reverse transcriptase PCR
- sequence cDNA
- align the cDNA sequences along the genomic sequence
RNA seq advantages over microarrays
- more accurate at quanitfying the amoung of each RNA
- superior at detecting RNA in low abundance
- shows exact boundries between exons and introns
- identifies 5’ and 3’ end of RNA transcripts
gene knockout collections allow
researchers to study gene function at the genomic level
recognition strategies
- sequence recognition
- pattern recognition
recognition strategies
- sequence recognition
- pattern recognition
search by signal
tries to locate an organization of known sequence elements that are normally found within a gene
search by content
tries to identify sequences that differ significantly from a random idstribution due to codon bias
open reading frame searching
looking for a region that does not contain any stop codons
homolous genes
are derived from the same ancestral gene
7 observations that show that a disease could be genetic
- disorder more likely to occur in genetic relative than general population
- identical twins share disease more than fraternal
- does not spread to individuals sharing an enviroment
- different populations tend to have different frequencies of the gene
- disease has an age of onset
- resembles a genetic disorder seen in other mammals
- correlation is observed between disease and a gene or chromosome alteration
personalized medicine
use of patients genotype and other things to treatment
concordance
degree to which a disease is inherited
concordance calculation
perceptage of twin pairs in which both the twins exhibit the disorder
identical twins = concordance is one
features of autosomal recessive inheritance
- an affected offspring have two unaffected parents
- when two unaffected hetrezyotes have children, ~25% have disease
- two affected peoples children have 100%
- occurs with the same frequency in both sexes
disorders that involve _ are typically autosomal recessive
loss of function in enzymes
50% of functional enzyme enough for healthy heterozygote
disorders that involve _ are typically autosomal recessive
loss of function in enzymes
50% of functional enzyme enough for healthy heterozygote
autosomal dominant inheritence features
- always have affected parent
- one affected parent have 50% affected offspring
- two affected hetrozygotes have 75% offspring affected
- same frequency in both sexes
- homozygouse more severly affected
three explanations for dominant disorders
- haploinsufficiency (50% not enough for non affected phenotype)
- gain of function mutation
- dominant negative mutation (mutant acts acts the wild type)
features of x linked recessive
- males more likely to show the trait
- mothers of affected males have brother or fathers who are affected
- the daughters of affected males will have 50% affected sons
x linked dominant inheritance features
- males more severly effected
- females show trait when it is lethal to males
- affected mothers have 50% passing the trait to daughters
locus heterogeneity
a disease can be caused by mutations in two or more different genes
microarrays in cancer
- can characterize tumors
- show the difference in cancers
- shows expression, cancer patients have more expression in certain type of cells that can show different types of cell cancer that presents the same
pharmacogenetics
the study or clinical testing of genetic variation that cuases different responses to drugs
characteristic of cancer
- clonal
- multistep process
- invasic and matastatic
clonal
cancer cells originate in a single cell
two types of cancer genes
oncogene
tumer supressor gene
oncogenes are _ mutations while tumor supressor genes are _ in cancer
gain of function, loss of function
four common types of changes that produces oncogenes
- missense mutations
- gene amplifications
- chromosomal translocations (philidel transolation = overexpression of BCR-ABL fusion protein)
- viral integration
oncogenes often encode…
- growth factor receptors
- intracellular signaling proteins
- transcription factors
cancer can affect Ras by
- decreasing activity of GTPase
- increase the rate of exchange of bound GDP for GTP
- Ras signalling pathway stays on longer than normal
Rb protein normally
prevents proliferation of cancer cells
* Rb is phosphorylated when cell is about to divide, lets go of E2F that activates genes for cell cycle progression
* when no Rb copies, E2F is always active
p53
- makes sure that cell doesnt divide w dna damage
- activates genes that promote DNA repair
- arrests cells division (stimulate p21 which inhibits cyclin/CDK complexes)
- activates apoptosis (activate caspases)
two categories of tumor supressor genes
- proteins that negatively regulate cell division (Rb regulates E2F)
- proteins that maintain genomes integrity
3 ways tumor supressor genes can be silenced
- mutation in gene itself (promoter, introduce stop codon)
- aneuploidy (chromosome loss)
- epigenetic changes
loss of hetrozygosity
loss of the functional copy of a gene
* dominant fashion inheritence
* point mutation in normal allelel
* chromosome carrying functional copy is lost
epigenetic changes relating to cancer
- DNA methylation
- covalent modification of histones
- chromatin remodeling
gene pool
all the alleles of every gene in a population
population
group of individuals of the same species that occupy the same region and can interbreed with each other
The alpha-globin gene in humans and beta-globin gene in humans can be described as
homologs.
paralogs.
members of a gene family.
all of the above
A DNA microarray is a slide that is dotted with
known sequences of DNA
If a computer program is designed to recognize a directional arrangement of bases, such as any inverted repeats in transposable elements, this is an example of
pattern recognition
For a rare inherited disease, the theoretical concordance for an autosomal dominant disease among fraternal twins is
50%
Gene X can exist as a normal allele and also as a dominant mutant allele that causes a disease. You purify protein X from a homozygous normal individual and also from an individual that is homozygous for the mutant allele. You have an assay to determine the function of protein X. When you assay 1 mg of the purified protein from a normal homozygote, the activity is 100%. When you mix together 0.5 mg of the normal protein and 0.5 mg of the mutant protein, the activity is 13%. Based on this experiment, why type of dominant mutant is this?
dominant negative
A pre-disposition to develop breast cancer due to a BRCA-1 mutation displays a _ in a pedigree.
dominant inheritance pattern
At the cellular level, the BRCA-1 mutation is .
recessive
The mutation in BRCA-1 that promotes cancer is a
loss-of-function mutation.
local population
smaller groups within populations
populations may change in
- size
- geographic location
- genetic composition
polymorphic
gene that exists as 2 or more alleles in a population
polymorphisms are often causes by
SNPs, a single base pair change in DNA
beta globin gene variations
SNP that causes sickle cell disease
deletion that eliminates function
allele frequency
number of allele in pop / total allele in pop
genotype frequency
number of indi w geneotype / total number of individuals
for monomorphic genes, the allele frequency
will be equal or close to 1
for polymorphic genes, the allele frequency
should add up to 1
conditions of HW equilibrium
- no new mutations
- no genetic drift
- no migration
- no natural selection
- random mating
chi squared test for HW equalibrium
if null hypothesis is not rejected, population in Eq
- if null hyothesis is rejected, population in disequil
microevolution
changes in a population’s gene pool from generation to generation
microevolution is driven by
list
- mutation
- random genetic drift
- migration
- natural selection
- non randoming mating
_ is the source of new genetic variation
mutations
_ change allele frequency the most from generation to generation
random genetic drift and natural selection
genetic drift
allele changes as a matter of chance
reproductive success
the ability to survive to reproductive age in a particular enviroment
natural selection acts on
phenotypes
which govern by genotype
atural selection acts on
phenotypes
which govern by genotype
principles of natural selection
- within a population there is allelic variation arising from various factors such as mutations
- some alleles may encode proteins that enhance an indiciduals survial or reproductive capacity
- idividuals with beneficial alles are more liley to reproduce
- allel frequencie s of many different genes may change through natural selection
darwinian fitness
the relative likelihood that a genotype will survive and contribute to the gene pool of the next generation
relative fitness
- gene with highest reproductive sucess is given fitness value of 1
- other ones are assigned vallues based on how many offspring they make / how many the highest one made
list four ways natural selection operates
- directional selection
- balancing selection
- disruptive selection
- stabalizing selection
directional selection
favors the extreme phenotype
* switches back in forth between high and low extremem in some populations
* frequency of AA: p2 x highest fitness value
* Aa: 2pq medium fitness
* aa: q2 times lowest fitness value
* add up to mean fitness of the population
calculate mean fitness if
A= 0.5
a = 0.5
AA indiv: 5
Aa: 4
aa: 1
= (0.52)(1) + (2)(0.5)(0.5)(0.8) + (0.52(0.2)
= 0.25 + 0.4 + 0.05
= 0.7
- to get frequency after one generation of direction selection, divide each term by 0.7
