Cell Physiology Test 1

cells use different compartments to do different processes in order to be efficient

reactions occur when particles randomly bump into each other. Conditions can be changed in order to make the encounters more or less likely

components are linked together to make things which can be broken down into their components to be recycled

different things have specific functions but can also perform more than one function simultaneously

processes follow a pathway that was determined by evolution and not necessarily because it was the best way to do it

determine the developmental plan, are highly conserved, and the position of the gene on the chromosome is similar to the point in the body plan it is coding for

GFP is always glowing while Luciferin only glows in the presence of oxygen

very slow, because a new polymerase needed to be added after every cycle due to the denaturation at high temperatures

DNA Polymerase that was isolated from thermus aquaticus at yellowstone national park. Brock was interested in Taq, Chien and Trella isolated Taq Polymerase, Mullins used it, and LaRoche patented it

• 1. Denaturation (95C) - separates the DNA 
• 2. Annealing (60C) - primers stick to complementary areas
• 3. Polymerization (70C) - Taq Polymerization adds the free nucleotides until it falls off the strands
• Ingredients: DNA, free nucleotides, primers, and Taq Polymerase

pure sample

enzyme that chews up and degrades RNA from the 3' end

• cDNA is more stable than mRNA. 
• mRNA has a 3' poly A tail so an oligo-dT primer (poly T tail) to bind to the mRNA in an RNA sample and then using reverse transcriptase we convert to cDNA

Consists of all the RNA that a cell had at the time it was killed, when it is converted to cDNA it is a cDNA library

• ligate Gs to one side and Ts to the other
• cut the DNA and the plasmid with the same restriction enzymes so that they are completely complementary and the cDNA can be added to the vector

a site that can be recognized by restriction enzymes so that you can make them do what you want

so that you can grow plasmids on ampicillin to ensure the survival of bacteria that has taken up the plasmid only.

bacteria will suck up DNA from the environment if they are stressed. We use the sos system so that we can force the cells to take up the plasmids

42 C and CaCl. These conditions kill most cells

putting the bacteria in a centrifuge and popping them open with chemicals and DNA. This process will produce so much DNA that it can be seen with the naked eye

more stable, less mistakes than Taq polymerase, more DNA, can accomplish more with a vector than a PCR product

missing 2 oxygens

• label dideoxy nucleotides with a color
• they randomly stop PCR and statistically if the products are sorted by size there should have been a dideoxy nucleotide at each bp
• the sequence is recognized and determined by a laser

expensive, slow, and overshadowed by new techniques

uses a semi conductor chip which has wells that capture chemical information and translate it to base pairs

to separate the molecules you are interested

Agarose gel is used to transfer Nucleic Acids because they are big and negatively charged

• Bigger molecules are at the top and the smaller ones are at the bottom because smaller molecules travel further
• Relative size is determined through the use of the ladder
• Brightness and thickness is proportional to the amount

A blot is a membrane that you transfer the target molecule to

The purpose of a southern blot is to determine if genes in a species are found in other animals by using a probe from one animal and DNA from another, copy number, and differences among genomes

Diffusion because the membrane is thinner (nitrocellulose paper) and the bands are on the surface of the gel

• Cleave the DNA with restriction enzymes
• run electrophoresis
• transfer the bands from the gel to the nitrocellulose paper via capillary action
• hybridize with labeled DNA or RNA probes

RNA

the size, presence of a sequence, and the amount of RNA

similar to southern blot except you use RNA and you incubate the membrane with a radio-labeled probe

Real Time PCR (qPCR) and chips

qPCR is used in order to determine how much mRNA is made in condition A vs. condition B

It doesnt tell us the size of the mRNA because only a small piece of cDNA can be amplified

It is easier than measuring the amount of protein that is made

extract DNA, run many cycles of PCR, DNA is detected while it is being made using a fluorescent probe that sits in the middle of the strand and gets knocked off by the polymerase, the probe doesn't fluoresce when it is knocked off

• X is the cycle number and Y is the amount of fluorescence 
• How soon the signal amplifies is proportional to how much you started with

it is more sensitive because you measure the data while it is happening and you can see if there are different starting amounts rather than just the ending amount like in PCR

it is so sensitive that you can detect a single molecule, draw a line across the middle of the curves, determine what cycle it crossed the threshold and then use a formula to determine how different the two conditions are

diagnostic tests for genetic and non-genetic diseases

An incorrect qPCR study

Separating proteins, transferring them to a blot, and then incubating them with an Ab

the size, the presence, and the amount of protein

• The gels used are usually acrylamide or resin based instead of agarose which are carbohydrate based
• Acrylamide gels have smaller pores and allow you to look at smaller things

the gel is transferred onto a membrane using electricity because the membrane is too thick to transfer by capillary action, an Ab is used against particular protein and then a second Ab that is labeled is used in order to see where the protein is

used for AIDs testing (test against the presence of Ab against HIV)

Proteins are coated with dodecylsulfate or soap becayse they do not have a uniform charge. the soap unfolds the protein and coats them with a uniform charge

Proteins are coated with soap (dodecylsulfate) because they do not have a uniform charge and the soap causes them to unfold and coats them with a uniform charge

Polyclonal Abs are more general and against multiple epitopes. Within a species. They are made in a limited supply through two antigen injections into an animal and then the harvesting of serum. The Ab cannot be remade when you run out of serum

Monoclonal Abs are more specific and are made against a single epitope. They are made by injecting an animal with Ag, harvesting spleen cells, fusing the cells with tumor cells and growing them on HAT media, then testing to find the cell that is against the correct Ab and harvesting it. These cells, B cell hybridomas, are immortal and you will have the Ab for forever

a primary Ab is against the thing you are trying to detect and is usually not labeled

Ab against the primary Ab and is usually labeled because you can make more of it while the primary Ab is in limited supply

A western blot of a tissue sample that is looking for protein concentrations

a northern blot that is searching for RNA and DNA in a tissue slice

modification of the genome without changing the actual base pairs via methylation and covalent modification

23 volunteers that did bike training on one leg only and submitted to muscle biopsies before and after in both legs.

• RNAseq was used in order to count the experimental myoglobin mRNA and the control myoglobin mRNA 
• It is better than a microarray

contraction, myofibril bundles, sarcomeres, regulation of glycolysis, regulation of adipose deposition

4000

Microarray is used to look at a bunch of DNA at the same time as well as compare samples by labeling the different treatments different colors.

Isoltate the total RNA from each sample, reverse transcribe each into cDNA, label the different treatments with different colors (green or red), hybridize to a glass slide with oligos that are stuck to the slide, the color will indicate where the cDNA sticks and then you can look up which gene it is

Just requires labeling.

RNAseq is similar to microarray but it is cheaper, more sensitive, and more quantitative.

collect RNA from your 2 conditions, make cDNA, make a library and sequence all of the cDNA (this will result in sequencing the same type of cDNA thousands of times), send the cDNA to a company who will send the data back to you

RNAseq requires a library and does not need labeling as a result. The library is made so tht you only have to use one primer to sequence everything.

The RNA transcriptase must be good at both short and rare or abundant and long sequences, and in order to publish your results you must use one of the RNA transcriptase molecules that is determined good.

you have so much data that you may not even know what the results are. There is likely to be about 30-40% of data that you do not understand

A developing field that analyzes mass quantities of data such as the results of RNAseq

A candidate gene is a gene that goes way up in a disease compared to the control

Knock outs or knock downs

A knock out experiment is when there is no expression of the gene of interest, there are traditional methods as well as CRISPR, you would typically raise the animal to an adult to see how the lack of gene effects everything, and a lot of times there is no obvious answer to what the gene actually does

A knockout experiment that uses RNA and the Cas9 enzyme

A strand of RNA is used as a guide which hybridizes to a specific spot in DNA which will lead to a double stranded break, the cell has repair mechanisms which repair the break and typically makes a mistake resulting in changes in the gene that can result in the termination of protein synthesis. The last step is to screen the organism to see if the mutation actually occured

Cas9 is an enzyme that is used in CRISPR, it comes from bacteria that recognize bacteriophage and chop them up

Big Mouse has no leptin production due to a lack of the leptin gene, if you connect its circulatory system to a normal mouse or give it leptin it will go to normal size

A knockdown experiment is when you reduce the amount of mRNA that is available to be translated

RNAi is a knockdown experiment that lasts for about 2-3 days

introduce mRNA that is complementary to the mRNA strand that you want to knock down, the mRNA will become double stranded and then will be degraded. Can be done in mammals and C.elegans

Place the RNA vector in bacteria and then feed it to the worms

Morpholino oligonucleotides bind to the RNA of interest and don't allow the translation of mRNA for about 4 days. Can be done in zebrafish

UTR (untranslated region), exons, and introns

Because components, such as introns, are missing

chromosomes must be unwound to be transcribed, long range regulatory elements, cis-regulatory elements (part of the DNA sequence which regulates how much you make), trans-regulatory elements (outside of the DNA that binds and regulates), and transcription factors (assemble on the promoter)

Isolate RNA, make cDNA, run PCR, label the PCR product, use the probe to screen a genomic library

Separation of charges that results from the pumping of protons across the membrane as electrons travel through the electron transport chain

The direction that the protons move

Double membrane bound organelles that were once free living single celled organisms that were engulfed by higher organisms

• Kidney bean shape (comes from the fact that they are fixed in EMS, actually more like a lava lamp)
• A cell has a specific number of mitochondria (cant be counted in a living cell because of fusion and fission)

Test for glycolytic enzymes, if they are not present, the sample is pure

Protein (as much as 90% by weight)

• Chloroplasts have 3 membranes instead of 2
• can use energy from light to move protons

Originally because they have mitochondria and they take oxygen from the environment at night. Now it is because of the potential for infection

The F1F0 channel has a different orientation. In chloroplasts it uses light to excite and move electrons. There is also a different terminal electron acceptor

Photorespiration is the process through which carbon dioxide is released instead of fixing the carbon into a 3-carbon molecule. It is catalyzed by rubisco which causes it to be inefficient as well as the low affinity for carbon dioxide

C4 plants are better at photorespiration because of compartmentalism.  They can concentrate carbon dioxide in one tissue and then move it to the tissues where photorespiration occurs

13 subunits, 10 by the nuclear genome and 3 by the mitochondrial genome

miss folded proteins will be placed in vessicles, the cell will die, and lesions result

The catalytic domain, protein rotates and the F1 domain changes shape allowing the addition of ADP and P to make ATP. It has 3 conformations: open, low, and tight. B subunits stabilize the head and keep it from moving

F0 is the channel domain, it is a half channel because the proton binds to Arg-210 causing a conformational change and the proton leaves on the other side.

Asp 61 on each C subunit binds protons.

a channel that destroys the proton gradient because it allows the passage of protons without the generation of ATP. These channels generate heat instead of storing energy in the form of ATP.

Infants have UCPs in their scapular fat which they use to regulate their body temperatures.

Sea slug that eats chloroplasts from algae and instead of digesting them keeps them intact and puts them on its mantle in order to photosynthesize and feed itself.