bio exam 2

Transgenic animals

animals that have copies of other animals genes.

Fluorophene tagging

often used for western blot

Fluorophores (which glow at certain frequencies) are conjugated’/attached to antibodies

antibody

Molecular cloning with gfp

-Clone a proteins cDNA adding GFP’s cDNA to the end, the fusing is called annealing

-so those proteins will glow, but only the ones made using that cdna

-genomic copy can be gfp tagged by the gfp being inserted into the cas9 so all proteins glow

parts of antibody

fab region= part of antibody that binds to pathogen

Fc region=constant region, its what immune cells recognize

foreign copy of gene vs native

exogenous vs endogenous gene

PCR

primers are about 20 nt loing

melt, anneal, extend

The DNA then doubles, then you do that for 30 cycles.

Restriction enzymes

recognizes 4-6bp sequence cuts both strands of dna (like through the double helix) w that sequence

from bacteria due to viruses

lab use of restriction enzymes

diagnostic digest, run dna restriction through gel

sequencing

reading out dna letters/base pair sequences

Gel electrophoresis

 DNA pulls to bottom with positive charge, if its shorter it will go faster

Phages

viruses that kill bacteria by insrtting their dna and taking over

cas9

enzyme that binds to a crRNA that has the sequence you want to cut, also binds to tracrRNA allowing it to bind and cut to sequence

the enzyme binds to a PAM site which is a sequence that only exists in that virus or sequence so it doesnt bind to bacterias dna that is where the cas9 is from

issue with cas9 editing/what the body often does to repair

Non-homologous end-joining
once the gene is spliced the cell just puts the normal/Non homologonous adelogonous ends back together

 can cause frame shift mutations, often cause major loss of function of protein

BUT researchers can add in a tempalte strand to try to get homologous directed repair to occur

Homology directed repair

where the chromosome is cut degredation occurs on either side on opposite parts of helix causing single stranded over hangs, one single stranded overhang dna binds to a copy of the correct dna, the dna is transcribed after that and then it goes back to old dna and an enzyme transcribes on tope of that

Reverse transcriptase

enzyme that makes dna copy of rna called a cdna,

hiv uses this by copying its genome which is in rna and putting into our cells, this is a retro virus

we use it for qRT PCR because rna is hard to work with since transient molecule

qRT PCR

pcr that copies rna by making it cDNA, finding out how much rna on plate,

flouresence increases with each copy, depending on how many cycles needed to get to an amount of flouresence you know how much rna you have

Ct(threshold cycle)= #doublings before a certain amount of fluorescence is achieved.

Fluorescent in situ hybridization (FISH),

 Shows where RNA is by probes that match sequences are labeled with certain colors.

Expression pattern is a map of the different rnas marked by color in organism.  

RNA sequencing

takes a sample and sequences rna by using cDNA, you learn how much rna there is/what is present

RNAi

viral dsRNA/double stranded RNA is cut up by a dicer when it enters cell

this cut up dsRNA is siRNAs

siRNAs go into a RISC complex which look for places that that strand is in mRNAs and chops them up

RNAi for research

 if you give the siRNA to a c elegan they will get rid of certain genes and the phenotypes will show what those genes effect

also can be used to help reduce or stop function in  gain of function mutations like cancer

Western blot

Western blot grinds up cells and extracts proteins then runs it on a gel, antobodies stick to protiens which makes them visible in gel

Beta actin is a loading control that shows the loading was done properly

p53

transcription factor that stops cell division when there is damage

RNA processing

 splicesomes that splice the introns out can leave in or leave out different exons to make different mRNAs

these different mRNas are called isoforms or spliceforms

Ubiquitanation

Ubiquitin is a polypeptide that a protein needs to be degraded by a proteasome.

  is added to proteins by ubiquitin ligase

location shift on western blot

Location shift on western blot usually indicates post transitional modification, because a phosphate group is heavier and changes where it is in the blot

Phosphorylations

phosphate groups are bulky and negatively charged, adding them to an amino acid can alter properties of a protein. It changes the confirmation of the protein into an active or inactive state. 

Kinase adds it

phosphatase adds phosphate

Post transnational regulation

change that occurs to protien after transcribed

Translation regulation

Eukaryotic initiation factions (eiFs) are proteins that control how fast a ribosome can be recruited to translate a mrna,

  if mRNAs are making proteins too fast eiFs can also shutdown protein synthesis

Protien regulators

bind to control elements/DNA regulatory elements

mutation in the protein can alter binding, dysregulation of gene expression 

motif

the dna sequence that the transcription factor binds to

Activator

transcription factors that help recruit rna polymerase, they bind to near promoter or to dna sequences enhancers

Repressors

transcription factors that block recruitment of RNA polymerase, they bind to silencers

control elements

like Silencers, enhancers, and promoters

dna sequences where transcription factors bind.

attaching control elements can trigger genes to be expressed or represes

Promoters

where the rna polymerase binds and lands

transcription factor

affect how the rna polymerase to binds to the promoter 

can be turned on by enviormental cues

myoD transcription factor

 induces expression of many muscle genes and transfection fibroblasts (connective tissue cells)

ginds to an enhancer and expresses multiple genes , the genes are kind of all over the place not necessarily in a set, it is similar to the operon though otherwise. 

Epigenetic regulation

affecting gene regulation before transcription

histone acetylation-weakens the gene attraction to histone, making it more accesible upregulating transcription

methylation- adding chemical tags on the dna, usually methyl groups on cytosine, usually makes it less acessbile to rna polymerase reducing transcription

set of all rna

transcriptome, varies between cells

set of protiens

protiome varies between cells

A niche

a  specialized environment within a tissue that nourishes and maintains stem cells

Outside the niche stem cells might die or differentiate

stem cells potential

totipotent-can make all cell types

Pleuripotent-can make all cells except a placenta

Multipotent-make cells within a cateogry

Unipotent-can make one kind of cell, is a stem cell can make another stem cell too 

Differentiated-has a job can replicate sometimes, sometimes differentiated cells are only replaced by stem cells not them dividing

Post mitotic- cant replicate

somatic cell 

cell that forms part of multicellular organism, not sperm or egg

epigenetic regulation affect on cell type

constrains what type of cell it will be because it changes what can be expressed

transcriptional regulation

Transcription factors control which rnas are made, creates a different pattern of gene expression controling what cell is expressed

sickle cell

mutation in alpha hemoglobin cells on the beta hemoglobin gene causing hbs cell

fetuses have gamma hemoblobin which cant really have the mutation so  Bc11a can be inserted to reactivate those cells but involves marrow transplant

agar and different cell types

soft generated neuron medium generated muscle and hard generated bone like

iPSCs/ induced pleuripotent stem cells

steps

take cells

reprogram using four transcription factors

allow to proliferate

differentiate

Ldr

ligand made in lab

often used on stem cells which gfp can bind to

dna stain

dapi

transmembrane receptors

Ligand gated ion channels/iontropic receptors

G protein coupled receptors/GPCRS

Enzyme linked receptors

kinase recpetor/phosphorylating receptor

 ligand triggers intracellular activity

sometimes triggers dimerization which causes molecues on thing to join

BMP

example of enzyme linked receptor

bone morphogenic protein= ligand

receptor=tyrosine kinase/phsophorylates tyrosine

kinase phosphorylates a transcription factor called Smad

then becomes pSmad which goes into nucleus and turn on bone genes 

Nuclear receptors

reside in cytoplasm

the ligand goes into the cytoplasm

when a ligand binds it goes into nucleus

Androgen insensitivity syndrome

 ligand of androgen receptor is testosterone

testosterone and other androgen hormones drives a development of a penis

mutation of the androgen receptor causes change in development of genitalia

Quorum sensing

bacteria that communicate how densely packed they are

cell signaling

used by all multicellular organisms

can trigger post transitional modificatios and trascription factor activity

cell division, neurotransmission, cell movement/cytoskeletal remodeling, apoptosis (programmed cell death form killer t cells) are examples

often occurs when kinase signals other kinase

Antagonist

a molecule that blocks that receptor

like narcan

Agonist

a artificial ligand that also activates that receptor

like morphine

Endogenous ligand

the molecule your body naturally uses as a ligand for the receptor

like endorphins to opiod receptor

parts of cell signaling

transmembrane protein that has a receptor domain on the outside of the cell where a ligand/effector binds

this can turn on intracellular second messenger which can eventually trigger the release of more effectors (which is called amplification) and other things

Different receptors may have the same ligand that lead to different responses.

kinases can be triggered in the same and opposite ways

cAMP example

cAMP=intracellular second messenger, activates protein kinase a (PKA)

PKA phosphorylates CREB

CREB is a transcription factor

extracellular first messenger

ligand

signal transduction

after receptor The signal is relayed, converted, and often amplified through a molecular cascade inside the cell.