Molecular Genetics Test 4

Human insulin production

make an expression vessel for insulin, take out insulin producing gene from human pancreas (based on mRNA -> reverse transcription -> cDNA0
put cDNA into bacterial plasmid, creating recombinant DNA
put recombinant dna into a bacterial cell
then put bacteria into fermentation tank allowing bacteria to multiply and produce human insulin in fermentation tank
extract and purify human insulin
Overall: using cloning technology to express the gene for human insulin and allow an expression vector to transcribe and translate it in a fermentation tank which is cleaner, less expensive, and don't have to kill pigs

Cloning an organism

took donor G0 cells, injected into enucleated oocyte (eggs)
electric pulse and then fusion of egg cells and donor G0 cells
This fusion cell is called renucleated oocyte

Cloning dolly by nuclear transfer

took mammary gland cells from a 6 year old ewe (female sheep)
Isolated and cultured cells
Isolated ovulated oocytes (removed the chromosomes)
end up with enucleated oocyte

Gene therapy

Example in animals - growth deficiency
lit/lit - dwarf mice
Growth hormone gene is present, but not expressed
GH gene placed downstream of an inducible promoter (methallothionein) - response to heavy metals (fed organisms heavy metals so they can translate the growth hormones)
inject plasmid into lit/lit egg into surrogate mother who will give birth to a transgenic baby
this can lead to a mouse of normal size

Transgenic Organism

an organism or cell whose genome has been altered by the introduction of foreign DNA sequences by artificial means

Transgenic Salmon

genes edited to make bigger salmon

Human gene therapy

Germ line gene therapy
Somatic cell therapy

Germ line gene therapy

introduce transgenic cells into the germ line(sex cells) as well as somatic cells
could result in cured parent, as well as offspring of the parent
must have targeted gene replacement
-gene disruption
-independent assortment of disease allele from introduced transgene

Somatic cell therapy

If treat a portion of somatic cells, should allow enough dominant alleles to mask recessive condition
1. Cells taken from diseased individual
2. Treated with transgene
3. Reintroduced into patient
Example: SCID
-Severe combined immunodeficiency
can treat humans with retrovirus that attack human cells
Bacterium carrying plasmid with cloned normal human ADA gene and genetically disable retrovirus.
Cloned ADA gene is incorporated into virus
T cells with disabled ADA gene isolated from SCID patient
Retrovirus infects T cells, transfers ADA gene to cells
Cells are grown in culture to ensure ADA gene is active
Genetically altered cells are reimplanted, produce dna

Ways to get transgene in

1. Disarmed retrovirus
-most only invade proliferating cells
2. Adenovirus
-Does not integrate, but can survive extrachromosomally
-primarily attacks nondividing cells
3. Human artificial chromosomes
-Introduced by Lipofectin, CellSqueeze

Knockout

Removing functional genes (adding nonfunctional ones)
DNA level
removing a working copy with nonfunctional version of that gene
used to suppress expression of gene
permanent and passed down through cells and offspring

Target gene recombination

transgenic organism that has nonfunctional version of a gene
If we want to knock the gene out or change the gene, we have gene A and we want to disrupt it, we could swap in our copy of disrupted gene

How do we make the mutant construct? In vitro mutagenesis
Accomplished by:

1. Site-directed mutagenesis
Creates mutations at specific sites in a gene whose wild type sequence is known
2. Also can use restriction sites, nuclease, or PCR to perform mutagenesis

Oligonucleotide-directed mutagenesis

Base-pair substitution
Insertion
Deletion

Base-pair substitution

Oligo binds to ssDNA
There's a mismatch between oligo and ssDNA
DS dna is created, then replication occurs and half have the mutant gene and half won'tt (replication will occur based on which ss dna is being replicated. If we have C and A matching, the next replication will have dna with CG and AT pairs

Insertion

Insert a piece of dna into oligo
when replications occur, half with have the inserts and half won't

Deletion

delete piece from oligo
when replications occur, half with have the deletion and half won't

Deletion

we can cut it with a restriction enzyme and let it aneal back to itself

Cassette replacement

with restriction enzyme we can cut out the good copy and put in the bad copy

Sets of deletions

Erosion with nucleases (deletion from one side, not in the middle)

pcr mutagenesis

long primer that has mutation site

Knockdown

mRNA/protein level
"knocks down" the production of a particular protein
does not require a transgenic organism

What do we do with transgene

we can knock in or knock out genes

knock in

adding genes

Knockout organism

we have gone in and changed an organisms genome so they have a nonfunctional copy of a gene that was functional

Transient knockdown

will stop the translation of mRNA, or the production of a particular protein

Gene knockdown methods

Antisense oligonucleotides
RNA interference (RNAi) - siRNA
ZFNs/TALENs - DBD and DNA cleaving domains
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

ZFNs

a protein that has dna binding domains and dna cleaving domains that will recognize specific sequences, and an exonuclease will come and cut sequences out of DNA
(cause deletions)

TALENs

same thing, repeat sequences that have a exonuclease chopping up a piece of dna (cause deletions)

Antisense RNA

inhibits translation of an mRNA
introduction of complementary ssRNA molecules (antisense rNA)
Delivered by chemical or mechanical means
Goal: to get RNA into cytoplasm, not nucleus
Usually made to the 3' or 5' UTR - more specific (3' and 5' UTR are use because many RNAs have similar coding regions, and we don't want those to degrade too
must bind together to inhibit translation
when antisense RNA binds to 3' UTR or 5' UTR the mRNA is degraded and translation is blocked
anytime our cells see dsRNA, they degrade it

RNAi

use of dsRNA homologous to specific sequence (since ssRNA degrades quickly use this instead)
double stranded RNA chopped up into 21-23 nucleotide sequences
Bound to RISC complex (RNA-induced silencing complex)
Guide RNA helps complex find homologous sequences
once bound, nuclease activity degrades mRNA

How to generate dsRNA

in vitro transcription -
stem-loop expression - have one promoter but a sequence that is able to fold over on itself and make itself double stranded
dual promoter - two promoters (one strand and then the other)

how RNAi works

we have our ds rna that we've made.
the dsRNA is going to be recognized as foreign.
Dicer enzyme comes in and chops up dsRNA and makes it single stranded and chops it up into pieces. When its done, it brings in RISC complex
RISC complex takes single stranded piece thats 21-23 nucleotides long and brings it to its complementary RNA sequence
then the nucleases chop everything up

Proteins in RISC complex

helicase
Rec A - Recruitment protein
Exonuclease
Endonuclease

RNAi Therapeutic Mechanism

Short interfering RNA (siRNA) designed to correspond to gene target (mRNA)
siRNA synthesized to be double stranded
RISC picks up siRNA, target mRNA is found and chopped up
silences production of protein we think would be bad

Example of RNAi

used RNA to look at a gene they thought was important
The gene on/expressed - no tumors
When used RNAi to chop up mRNA of this gene - more tumors

CRISPR

Recognition sequence of RNA that will recognize a specific dna. you can use it to make a knockdown or you can go and edit the genes
cuts pieces of the DNA out and can incorporate it into another DNA
gene editing and cat cut genes out
CRISPR - Cas9 gene therapy: have a mutation, we have a targeting piece of RNA to recognize piece of DNA so we can cut the DNA out and the correct sequence would be put in with no mutation

C. elegans as model organism

Introduced in 1963
Free-living, non-parasitic nematode (worm)
small, easy to rear, fed E. coli, short lifespan, lots of offspring
transparent (can see whats going on inside them)
Haploid number is 6, 97 million bp genome (relatively small genome)
Developmental fate of every cell has been mapped. They can track the fate of every cell (959 cells in hermaphrodites, 1031 cells in males)
Overall: cheap to feed, can make all sorts of offspring, entire genome mapped, they know exactly where all the SNPs are in the genome and the fate of every cell
Hibernation state called dower is studied as well

Zebrafish as a model organism

small tropical fish
hundreds of known mutants
easy to rear
transparent embryos
an regenerate tissue (a lot of research done in regeneration and development)
1.7 billion bases
47 fold coverage (they've sequenced through the entire genome at least 47 times)
genome is known very well
5 million known SNPs (you can make a SNP chip with all 5 million known SNPs and look for mutations)

Tol2 transposon

Tol 2 ends: regions that are going to be insertion sequences into the particular genome of interest
-can use this to get a green fluorescent protein into an organ
can look for expression of particular genes

Zebrafish used as biosensor system

Ex: estrogen gets into waterways
-make a biosensor system out of zebrafish
took an estrogen inducible promoter thats controlling the expression of green fluorescent protein
if there's green fluorescent protein, you know there's estrogen in the water

Transgenic organisms: Ways to get DNA into cells

1. Transformation
2. Virus
3. Micro injection
4. Projectile gun

Transformation

-have a cell competent to take up DNA and then you usually heat, shock the cell and chemically treat it so there will be pores forming in the membrane so things can be transformed in

Virus

will attack a bacterial cell and put in your construct

Micro injection

Long glass needles, stick them into the cell and inject whatever you want in

Projectile gun

bead-like bullets with DNA coated around them, shoot them into the cell. use this when cells have a cell wall so it won't destroy them unlike an animal cell

Mammals

create morula with cell division. then eventually a blastula will form

Morula

solid ball of cells

Blastula

hollow ball of cells

Flies

divide their nuclei multiple times before they divide the cells
this is called a multi nuclei syncytium

Transgenic flies

easy to make a transgenic construct and put it into the multi nuclei syncytium
there's an anterior and posterior pole of the multi nuclei syncytium. We know the posterior cells are to become germline cells and can inject DNA into the posterior end at the right time
then cell membranes begin to surround the nuclei '
we try to get the construct into the germline and then breed that organism and try and make a true transgenic organism

P element

one of the transposable elements used in drosophila. we have a bacteria with a DNA segment of interest and P elements on each side and inject it into the posterior end of the multi nuclei syncytium

First attempt: DNA microinjection to make transgenic organism

1. transfer of a desired gene construct into the pronucleus of a reproductive cell
2. the manipulated cell must be cultured in vitro
3. transferred to the recipient female
not efficient: not many pigs born with desired gene
in mammals it didnt work (injecting DNA into eggs)

Embryonic stem cells transgenics

can be recovered from different tissues, and these embryonic cells can be placed into developing blastula in order to incorporate themselves in (instead of eggs cells, embryonic stem cells)
Micropipette/microinject the construct DNA into the nucleus of an embryonic stem cell

How we create a transgenic mouse

Useful bc you can see what happens when you knock in or knock out genes
We have a particular gene, we want to knockout a gene of interest we want to interrupt the gene to not produce the proper protein
-incorporated into the genome of a host, so it will never be able to produce that protein
The vector: split an exon and put a big cassette of DNA, interrupting an exon
The cassette has resistance in the antibiotic neomycin and then a long stretch of stop codons. The cells will never produce that protein (premature stop)
A tyrosine kinase gene is put into the vector as well
we take stem cells from a brown coated mouse (we use this because it is the dominant color)

3 things can happen when we insert

1. never inserts, just gets lost, unchanged chromosome in cell, DNA is still the same, nothing knocked out, these cells have no resistance to neomycin
2. Targeted insert: get the vector put in exactly where its supposed to (in the host chromosome)
crossing over occurs where the sequences are homologous to each other
only 2 flanking regions: exon 2 and cassette
only cassette and exon 2 should be put in
3. Ectopic or random insertion: inserts itself in the wrong place, but doesn't interrupt the gene of interest
the whole vector gets put into the chromosome with this insertion usually
-will be neomycin resistant but will also have the tyrosine kinase

We only want the targeted insertion

plate the embryonic stem cells with neomycin analog (kills off cells that don't have neomycin resistance and only want ones will neomycin resistance to live) and ganciclovir
Neomycin analog: select against cells with no insertion
Ganciclovir activates the tyrosine kinase to start apoptotic cascade, prograaminf cells to die
Ganciclovir: selection against cells that have tyrosine kinase

Brown mice

We take our embryonic stem cells from brown mouse and inject them into a blastula from a black female (recessive)
Take early embryo that has all cells that code for a balck coat except for the embryonic stem cells we stuck in which will give rise to brown coat and also have the trans gene
then put the embryo in surrogate (black) mother and look for any mice that are chimeric mice (that have some brown fur)

Breeding

We take our chimeric mouse that has two cell types, recessive from black coated mouse and dominant from brown coated mouse
Take a male chimeric mouse and cross him with black female(that can only pass black coated alleles)
The sperm can either come from the original black coated mouse cells (can only pass a black coat allele and the mom could pass one too, ending uo with a black coated offspring that doesn't have the transgene.
If embryonic stem cells that came from brown mouse become incorporated into the gametes and that fertilizes the egg, we end up with heterozygote. Brown is dominant so the mice will be brown
We can still end up with a brown coated mouse with no transgene
Then we genotype them to find the brown mice with the transgene
But this doesn't give us a full transgenic organism b/c they're only heterozygous. we need both copies of transgene to be a true transgenic
We cross litter mates that are brown and have the transgene, and 25% will be homozygous recessive (true transgenic)
We can make transgenic mice and use it to see what happens

Uses of transgenes

Can put a transgene for bioluminescenece, made bioluminescent cats and rats
Obese mouse, unable to produce leptin and normal mouse
Leptin is produced after we eat so we feel full
Genetically modified cows that produce human milk since children don't do well with cow milk
Genetically altered fish made to be larger
Transgenic cows producing myelin basic protein

Normal/Non-cancer cells

Death cues from another cell will turn on programmed cell death cascade and will undergo cell death
Growth inhibition cues will lead to division checkpoints being blocked and no cell division will occur
If you don't get survival cues the cell may not survive
If there's no proliferation cues, cells do not divide

Cancer cells

ignores death cues
produces its own survival cues and proliferation cues and growth inhibition cues are ignored
leads to uncontrolled survival and proliferation

Sporadic cancer

majority of cancers are sporadic, meaning that mutations have occurred in the genes due to environmental influence, not necessarily through germline

Familial cancer

minority of cancers are familial where you can actually follow them through a particular family

Mitosis

Between mitosis and synthesis is the G1 phase of the cell cycle
M(mitosis) - cell monitors spindle formation and attachment to kinetochores
G1 - cell has to have the correct DNA before going into the DNA replication phase in S
S - replication phase
G2 - cell monitors DNA synthesis and DNA damage

cyclins

paired up with CDKs (cyclin dependent kinase)
identifies target and recruits in the CDK to phosphorylate that target

CDKs

a cyclin dependent on a kinase
if cdks are put in a tube with cyclins, they will phosphorylate anything in front of them

kinase

kinases phosphorylate another protein

G1

a series of phosphorylation events that occur
has to do with two other proteins: Rb and E2F

Reinoblastoma (Rb) and E2F

When Rb is bound to E2F it prevents it from becoming activated and going to the nucleus and doing it's job
Rb is sequestering E2F in the cytoplasm
As a complex/dimer and as long as Rb is bound to E2F, E2F can't go into the nucleus

Middle of G1: cyclin D and cdk4

in the middle of G1, cyclin D and cdk 4 become active. cyclin D will find Rb and cdk4 will phosphorylate Rb on specific residues. Then, cyclin D and cdk 4 gets degraded

cyclin E and cdk 2

become activated after cyclin d and cdk 4 gets degraded. cyclin E targets retinoblastoma and cdk 2 phosphorylates Rb on other residues putting Rb in a hyperphosphorylated state

End of G1

Rb will release E2F to go into the nucleus and start transcribing genes that are necessary for proteins in S phase (replication) like DNA Pol, PCNA, RNR

When cells recognize it has mutations

if cyclin D and cdk 4 don't phosphorylate Rb, it stops the cell mid G1 phase
If cyclin E and cdk 2 don't phosphorylate Rb, it stops the cell from going into S phase

CKIs

cyclin dependent kinase inhibitors
they inhibit cyclin dependent kinase
proteins that come in and bind to cyclins and cdks to prevent them from doing their job
can function any time in the cell cycle

p53

CKI, but its actually a transcription factor that turns on true CKIs
when p53 gets activated as a transcription factor it turns on p21 and p21 will bind to cyclin and cdks to prevent it from functioning

Cell cycle and cyclins/cdks

cyclin D/cdk 4 = middle of G1
cyclin E/ cdk 2 = end of G1, beginning of S
cyclin A / cdk 2 = drives the cell through S phase
cyclin B/ cdk 1 = middle of G2, activated through a dephosphorylation. cdc25 phosphatase removes phosphate groups from cyclin B and cdk 1, starts the process of mitosis

Tumors are classified according to tissue of origin

Carinomas from epithelial cells
Leukemias and lymphomas from blood cell precursors
Major problem
Every tumor type is individual and no single treatment exists to cure all cancers

Tumor formation

Benign tumor begins when cells continuously proliferate
Due to mutations in cell cycle regulatory genes
Different genes that may be mutated
1. Signal genes
2. Proliferation genes
3. Cell cycle checkpoint genes

Tumorogenesis

Made up of cells (clones) descended from one cell
Mainly result from exposure to mutagens, not through the germ line
Rates of cancer incidence varies die to location of population
Depending on where you live there's going to be a common % of individuals with cancer based on location of population
Immigrants begin to show the same cancer rates as indigenous people
Tumors usually develop over time

Oncogenes

Mutant alleles that act dominantly to promote proliferation. Can be heterozygous for the condition
even if you have one copy of the mutant allele it is going to act dominantly to allow proliferation to occur

Proto-oncogene

non mutant precursor to oncogene
only cause cancer when over expressed or amplified
Ex. cyclin d works mid G1 with cdk 4 to phosphorylate Rb. There are some mutations that can prevent it from being degraded so its always active. this would cause hyperphosporylation of Rb which would lead to E2F being let go into nucleus and transcribing proteins for replication (S phase)

Mutant tumor-suppressor genes

mutant alleles that are recessive. Both copies must be mutant to make the cell abnormal. CKIs bc even if you have one mutated copy, CKIs can still be expressed
cause cancer when deleted, not working anymore

How are proto-oncogenes activated

involves a gain of function
1. Activation by amplification
-cells contain several copies of structurally normal oncogenes
2. Activation by point mutation
RAS family members
3. Activation by translocation
puts oncogene into transcriptionally active region

RAS

There can be a single point mutation in ras wild type DNA turning a Gly into a Val
Regularly ras can be phosphorylated to be activated when needed and dephosphorylated to be inactivated when not needed
This causes Ras to get turned on and it can never be inactivated. It keeps the intracellular signaling cascade going which tells the cell to continuously divide

Viral-mediated cancer

can be caused by viruses
tumor viruses transform normal cells into cancer cells
large number are retroviruses - utilize reverse transcriptase
protooncogenes can become oncogenes if come under the control of viral promoters
viruses can incorporate a promoter sequence that makes constitutive cyclin to be expressed all the time which can lead to tumor formation
cyclin K - can't be stopped by CKIs so its always turned on
some viruses contain oncogenes
Ex. Human papilloma virus (HPV)
-Some HPVs carry at least two oncogenes
E6: causes degredation of p53 (transcription factor that turns on genes for CKIs)
e7: binds and activates Rb (bound to E2F) binds to Rb so it can never hold E2F which turns on genes to be pushed into replication (better change of mutations getting through)

Malignant tumor must acquire

1. Independence from external growth factor
2. Insensitivity to anti-growth signals
3. Avoidance of apoptosis
4. Capability to proliferate indefinitely
5. Capability for sustained angiogenesis
-once these cells transform from benign tumors to malignant tumors, they start a network of surrounding themselves with blood vessels and steal nutrients from the blood and become oxygenated. Angiogenesis - formation of blood vessel
6. Capability to invade tissues and metastasis
-Malignant tumors are capable of invading other tissues in the process of metastasis (they transform cells; it can break off from one tissue and start growing in another

Apoptosis

Step of programmed cell death (PCD)
Cellular suicide (potentially getting a signal but programming itself to die)
Used by multicellular organisms to eliminate damaged cells
Driven by intracellular signaling
-Proliferation and apoptosis are not always bad
Mitochondria are ruptured (or not*)
Release of cytochrome c (part of Electron transport system)
Activation of caspases (or not*)
Chromatin condensation and membrane blebbing
-DNA chopped up into little pieces and cell membrane is going to be very ragged

Apoptosis process

Normal cell
then apoptosis initiated
Mitochondria broken open
DNA becomes punctate (small) and gets chopped into little pieces (cells can't live if their DNA is chopped up into little pieces)
The cell shrinks
Membrane gets beat up
Scavenger cells pick the pieces up

Alzheimers

Healthy brain - full cerebrum, aren't many big gaps
Brain with Alzheimers
-with a lot of gaps in the brain, a lot of apoptosis

Parkinsons disease

Dopaminergic neurons that die off via apoptosis

Cellular events in NGF deprivation-induced apoptosis

can take cells from an individual and then spot those cells down on a plate
the neurons will just grow out and form ganglia
In the presence of NGF the cells will grow happy and healthy and will grow a miniature brain inside the dish
If NGF is withdrew, the cells will die via apoptosis and we can see what goes on in the progression of apoptosis
A burst of oxidative stress 3 hours after NGF is removed (reactive oxygen species can trigger cells to do things)
-drives the cell into apoptosis
JNK (jun kinase activity which leads to c-jun (TF) induction)
12-15 mitochondria rupture and cytochrome c is released
then you get caspase activation, chromatin condensation and DNA fragmentation

Caspases

very important in apoptosis
proteins that degrade other proteins
cysteine-aspartic proteases
when activated, cleave target protein
Normally in inactive state (zymogen)
-must be cleaved to become active
-Initiator caspases 2, 8, 9, 10 that cleave zymogen into fragments
-Fragments come together to form active executioner caspase which leads to
1. Inactivation of DNA endonuclease sequestering protein
-> leads to DNA fragmentation
2. Activation of actin-cleaving protein
-> leads toLoss of normal cell shape
3. Other targets
-> leads to breakdown of organelles, fragmentation of cells
Effector (Active executioner caspase) - 3, 6, 7

Intrinsic Pathway

slower
cellular stress occurs (Ex removal of NGF)
Then there's mitochondrial dysfunction (spill out contents)
Bax and tBid are proteins that lead to mitochondrial dysfuntion (pro-apoptotic proteins)
Bcl-2 are anti-apoptotic proteins, this blocks mitochondrial dysfunction
cytochrome c and Apaf-1 gets released, which activates initiator caspase 9 which cleaves our effector caspase 3 which leads to apoptosis (24-48 hrs)

Extrinsic Pathway

Have receptors with death domains
A ligand comes and turns on death domain containing proteins which turn on a death complex which immediately activates caspase 8(initiator) which then cleaves caspase 3 (effector) to undergo apoptosis
Ex: death ligand called Fas
binds to a receptor called FADD
Fas binds to FADD and immediately turns on the death complex which activates apoptosis in the same extrinsic pathway

Caspase-independent apoptosis

Apoptosis-inducing factor (AIF)
Mitochondrial protein on X chromosome (sits in membrane of mitochondria)
Triggers chromatin condensation and DNA degradation
Multipotential mediator (has many jobs)
-Respiration
-Redox reactions

Intercellular signaling

Cell-cell communication (one cell produces a signal, the other cells going in to respond)
Ligand released by one cell binds to specific receptor on another cell
Triggers intracellular signaling in receptor cell
Two examples
1. Endocrine: Use of hormones as long-range signal molecules through circulatory system (small molecules)
Ex: a region in the brain releases a hormone and it goes through the body and any cell with the receptor for that hormone will respond
2. Paracrine: Short range protein ligands. Don't use circulatory system
Ex: important during development
One cell in the vicinity, maybe next to the cell producing a hormone or secreting something and nearby cells will respond