Mark is in trouble

Part I

1. What types of physiological problems do humans encounter at high altitudes?

 Headaches, dizziness/lightheadedness, shortness of breath, fatigue

2. What symptoms did the climbers exhibit that might be related to altitude? Explain. 

Headache, deep breathing, fatigue. Shortness of breath would be caused by the lack of pressure of oxygen in high altitudes which leads to the climbers to breath deeper. Fatigue is caused by the lack of oxygen as not enough oxygen is taken in (compared to sea level) to energize the cells. Headaches may also be caused by the lack of oxygen that is going to the brains.

3. Compare the air at 18,000 feet (atmospheric pressure 280 mm Hg) to the air at sea level (760 mm Hg). What specific changes in the primary atmospheric gases (nitrogen, oxygen, carbon dioxide) might occur? Are they significant? 

The composition of earth's atmosphere is 78% nitrogen, 21% oxygen and 1% other gases (including carbon dioxide). There is less pressure at 18 thousand feet than there is at sea level, which means that the density of oxygen molecules is less and therefore would mean the amount of oxygen inhaled by a human being at 18 thousand feet would be lower than one at sea level. (Air pressure in atmosphere is supposed to be greater than the pressure inside the lungs so that the air would "go into" the lungs)

4. What is the specific pulmonary response to high altitude? [Assume you are considering a subject at rest.] 

Lowered oxygen density in the atmosphere means less oxygen intake which means that the heart will pump harder to get more blood to pump throughout the body to try and catch up to the normal amount of oxygen that would be distributed at sea level.

5. How will this response affect overall blood gases? What about oxygen loading and unloading from hemoglobin? Explain how you arrived at your conclusions. 

There will be less oxygen binding to the hemoglobin in the blood as there is less oxygen in the atmosphere. There will be a higher affinity of RBC for oxygen

6. After breathing at altitude for a few days, the body normally begins producing more 2,3-DPG. What is the significance of this change? How will it affect the pulmonary changes observed?

2,3 DPG helps decreases the affinity of RBC for oxygen which means that the body will be able to increase the amount of oxygen distributed to the tissues as less would stick to the RBC. The person will be less fatigued, have a lower heart rate and less headaches

Part II

1. What physiological changes is Emily referring to (above) that will occur when someone lives at altitude for an extended period?

The body learns to increase production of erythrocytes, which increases oxygen in blood as there are more RBCs.

2. How are these changes advantageous? 

Cells in the body will receive more oxygen

3. What is the specific physiological pathway that results in the changes described?

Kidney detects low oxygen level and secrets erythropoietin, stimulates RBC production in bone marrow, more RBC = More hemoglobin = more oxygen in blood = more oxygen to tissues

Part III

1. How would the oxygen and Gammow bag help Mark? 

Oxygen and Gammow bag will increase the amount of oxygen in Mark. The bag increases the atmospheric pressure, which leads to less strenuous breathing because more air is going into the lungs.

2. If you were a member of the medical team examining Mark, what types of tests would you run? Why? [Try to focus on what types of things you would like to measure, whether or not you know of a possible test for them.] 

Blood sample, see concentration of oxygen/CO2 and pH levels

3. What types of results do you expect to find? Explain your reasoning.

Lower oxygen/CO2 levels in blood compared to sea level (lower pH) because there is less oxygen at 18 thousand feet than at sea level. So the body is trying to play "catch up" to maintain the distribution of oxygen but cannot.

Hominid Evolution and Carbon Dating

Hominid Evolution

Ardipithecus ramidus (4.4 million years ago)

-divergent large toe with a rigid foot

-pelvis tree climbing and bipedal activity

-ape ancestor not chimpanzee like

-canine teeth = same size male and female

-wooded environment

-3 ft 11 in, 110 lbs

Australopithecus afarensis (2.95-3.85 million years ago)

-ape and human characteristics

-apelike features:

face proportions 

braincase (small brain)

strong arms with curved fingers (climbing trees)

-human features:

small canine teeth

body stood on two legs

-could live on trees and ground

-males (4ft 11in, 92 lbs)

-females (3ft 5in, 64 lbs)

Australopithecus africanus (2.1-3.3 million years ago)

-rounder cranium w/ larger brain and smaller teeth

-apelike:

long arms

sloping face that juts out (pronounced jaw)

-pelvis, femur, footbones: walked bipedally

-shoulder and hand: climbing

-males (4ft 6in, 90 lbs)

-females (3ft 9in, 66 lbs)

Homo habilis (1.4-2.4 million years ago)

-larger braincase

-smaller face and teeth

-ape like features: long arms & prognathic face

-average (3ft 4in- 4ft 5in, 70 lbs)

Homo erectus (143k-1.89mil years ago)

-more adapted to the ground

shorter arms

long legs

=walk and run more

-average (4ft 9in - 6ft 1in, 88 - 150lbs)

Homo neanderthalensis (28-200 thousand years ago

-closest extinct human relative

-skull features

large middle part of face

angle cheek bones

huge nose for different air (humidifying and warming)

-shorter stockier bodies (cold environments)

-brain same size sometimes larger

males (5ft 5in, 143 lbs)

females (5ft 1in, 119lbs)

Homo sapiens (200,000 to present)

-large brains

-thin walled high vaulted skull

-flat almost vertical forehead

-smaller teeth

Summary

-The braincase gets larger incrementally

-Generally get taller and heavier

-Shorter arms and longer legs

-Nose smaller

-Smaller teeth

-Became only bipedal

-These traits are meant to suit us to live on the ground

http://humanorigins.si.edu/resources/intro-human-evolution

Carbon dating

Carbon dating is used to determine the age of fossils and requires the use of the radioactive isotope carbon-14. Carbon-14 is produced by cosmic ray protons which blast nuclei in the upper atmosphere, thus producing neutrons and bombard nitrogen creating carbon-14. This occurs at a rate which is constant, therefore we can use radioactive emissions of once-living matter and compare it to living ones. The results allows us to make a measurement of the time that passed.

Radioactive half-life is the time for half the radioactive nuclei to go through radioactive decay. Unstable radioisotopes decay into more stable forms (C-14 decays to C-12, which is stable.) 

Living organisms constantly exchange carbon with the atmosphere in the form of CO2; this results with the organisms to have nearly the same ratio of C-14 to C-12 with the atmosphere. However when the organism dies, it beings to undergo radioactive decay in which C-14 decays into a more stable C-14. This can be used to measure the amount of time an organism had been dead for.

Carbon dating is used for younger fossils because of the difference in half-life.

C-14 has a half-life of 5700 years which is useful for fossils that are 1000 to 10000 years old, whereas potassium (K40) has a half life of around 1.3 billion years therefore allowing it to be used for much older fossils.

Abiogenesis and Panspermia

The abiogenesis theory states that life came originated from simple organic compounds that were created on the earth from the pre-biotic soup that existed. However on the other hand, the panspermia theory states that life on Earth originated from comets and asteroids carrying bacterial life from the universe.

The Urey-Miller experiment provides support that the occurrence of abiogenesis is possible. The Urey-Miller experiment aimed to create organic compounds via a system that simulated similar conditions of pre-biotic Earth. The simulation consisted of a recirculating system with heated water (as the ocean), a chamber with electrical charges (as the atmosphere with lightning), and a condenser for gases in the 'atmosphere 'chamber to dissolve in the water. These chambers consisted of ammonia, nitrogen, methane, hydrogen gas, and carbon dioxide. After a while, the experiment's result showed simple organic molecules in the 'atmosphere' chamber, whereas amino acids and other organic acids were found in the 'ocean' chamber.

However, it is still possible for the panspermia to be valid because abiogenesis may have occurred on a different planet and comets may have transported the products to Earth while abiogenesis was occurring on Earth.

Scientists think the RNA replication or metabolism occurred first before protobiont cells began to form in micro environments for many reasons.

Scientists think RNA replication occurred first because RNA have the ability to replicate themselves as well as catalyzing reactions. RNA also have the ability to attract and link amino acids into proteins, which are the building blocks of life. Furthermore, scientists believe that RNA were able to persist because of the clay from volcanic ashes would cover it from harmful UV rays.

Scientists also argue that metabolism occurred before the protobiont cells began to form because they believe that the Earth may have started out with the first living thing on Earth as a primitive metabolic life sustaining itself with a series of reactions with CO2 or CO catalyzed by metal sulfide.

Protobionts are thought to be precursors to prokaryotic cells. They consist of abiotically produced organic molecules surrounded by a membrane like structure. Protobionts have characteristics that are related with life such as simple reproduction, metabolism, excitability, and the maintenance of itself to survive in its environment (with the help of its membrane). Due to this, many scientists believe RNA replication and metabolism occurred before protobiont cells began to form. RNA is needed for the protobiont cells to execute simple reproduction and metabolism is needed so it can energize itself to survive. 

However, whether RNA replication or metabolism may have been the very first occurrences of life, both the abiogenesis and panspermia theories still have validity because organic compounds could have still been created on Earth or survived on a comet traveling to Earth to create protobiont cells.

Pre-lab

Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase.

Gene transfer involving plasmids are usually used to alter the circular DNA molecules called plasmids that are usually found in plasmids. 

Stage 1: Obtaining a gene to transfer

-Restriction enzymes are put in the same container as the DNA strands

-During this stage, restriction enzymes are used to cut the targeted gene that is to be transferred from the DNA of an organism to the plasmid and create 'sticky ends'

Stage 2: Preparing a vector for the transferred gene

-Restriction enzymes are also put in the same container as the plasmids

-The same restriction enzymes that had cut the targeted gene from the DNA strand is used to create a cut in the plasmid, creating 'sticky ends' complementary to the DNA fragments from an organism

Stage 3: Recombinant DNA

-DNA ligase catalyzes the reaction and reconnects the cleaved plasmids with the DNA strands by creating covalent bonds

Stage 4: Isolation of transformed cells

-The recombinant DNA is then inserted into the host cell

-The cells that are transformed to contain the recombinant DNA are separated from untransformed cells

Crime Scene Investigation

1) Explain the content of the human genome: % protein encoding, % regulatory, % STRs AND distinguish locus from gene from alleles

-There is less than 2% protein encoding (code for proteins) in the human genome, around 3% STRs (short sequences of non-coding DNA), and around 8% regulatory genes that control gene expression

http://upload.wikimedia.org/wikipedia/commons/thumb/6/64/Components_of_the_Human_Genome.jpg/370px-Components_of_the_Human_Genome.jpg

 -A locus is the position of a gene on a chromosome

-A gene is the specific part of DNA base pairs on a chromosome that code for a phenotype

-An allele is a different type of gene in the same locus

http://www.csulb.edu/~kmacd/361-6-Ch1_files/allele.jpg

2) Explain the use of STRs in profiling to demonstrate the rarity of sharing all/most/many STRs

STRs (short tandem repeats) are used in DNA profiling because they can be highly polymorphic, which means there are many varieties of it. Therefore, STRs are very unique to each individual and therefore it can be used to discriminate between individuals esp. at a crime scene. 

3) State and describe the evidence of the gel to identify the suspect at the crime scene by referring the alleles found at the crime scene and the allele of the suspect(s)

-On the gel, the second column is the DNA found in the crime scene, which is subsequently followed by Suspect A in column three, Suspect B, Suspect C and Suspect D in the next three columns.

-As you can see below, the DNA found at the crime scene is exactly in line with Suspect C of the fourth column. This is because the alleles (one blue dash is an allele) exactly correspond

DNA Profiling

Write a brief description of the difference between RFLP and STRs

-RFLP (restriction fragment length polymorphism)- it indicates a difference between samples of homologous DNA molecules via restriction enzymes

-STRs (short tandem repeats)- they consist of short repeated sequences of DNA (2-8 nucleotides) and are located on non-coding regions of chromosomes. STRs are also polymorphic, which means that there are nine or more different types of alleles that can be detected.

-The STR and RFLP methods differ in DNA profiling and the STR has more advantages, which is why it is preferred today. While the STR only needs a very small amount of DNA to run, the RFLP does not as it requires a very large sample of DNA to work. Therefore, with a small amount of DNA, using the STR method reduces the likelihood of destroying sample due to the utilization of PCR. On the other hand, the RFLP method destroys the cell after it is completed. Furthermore, the STR method takes only around a couple hours to complete whereas the RFLP method could take weeks. STR is also more accurate than RFLP because STR targets specific sites unlike RFLP.

How many STRs does the FBI use to analyze?

-They use 13 different STRs, with repeats of four or five nucleotides, plus sex markers

Describe how PCR is used in DNA profiling

-PCR is used to amplify the specific regions of the DNA (STRs) and is therefore used to ultimately detect differences between STRs. During PCR, DNA fragments, with primers, are heated and cooled to activate primers and DNA polymerase in copying the fragments over and over again until the amount of copied fragments are "amplified"

Describe how electrophoresis is used in DNA profiling

-Electrophoresis is used to separate different DNA fragments (which were amplified with PCR) by putting the fragments onto polyacrylamide gel. The polyacrylamide gel in PCR separates different DNA fragments by size and the smaller the fragment, the further it travels through the gel.

Explain how the probabilities of having a specific set of STRs is extremely unlikely. Therefore if yours is present at a crime scene, what does that mean?

-The probability of having a specific set of STRs is extremely unlikely because one in every 10^-100 quadrillion share the same STR profile. The number is greater than the number of humans that ever lived, therefore each STR profile is specific to one person. If my STR is present at a crime scene, this means that you were definitely at the crime scene but does not guarantee any more than that.

Recombinant DNA Techniques

Extracting DNA

Reasons to Collect DNA

-Genetic testing

-Body identification

-Analysis of forensic information

Outline and explain the purpose of: swabbing, lysis buffer, centrifugation, and isopropyl alcohol

-Swabbing is the process of collecting a sample of cells. An example of this is swabbing the inside of somebody’s mouth to collect cells for DNA extraction.

-The lysis buffer contains detergent and enzyme proteinase K and separates the DNA from the cell. The detergent breaks down the cell membrane and nuclear membrane so that the cells would burst open and release the DNA. The enzyme cuts down the histone proteins, which the DNA is wrapped around, and therefore frees the DNA.

-Centrifugation causes the remaining debris and protein to sink to the bottom of the test tube while the DNA remains distributed in the liquid. This thus separates the DNA from any debris and protein, which may interfere with analysis.

-Centrifugation can also be used after isopropyl alcohol is mixed with the DNA so that the DNA would sink to the bottom of the tube, allowing the DNA to be isolated.

-Isopropyl alcohol concentrates the DNA together so that it is visible to the naked eye. This is because DNA is not soluble in isopropyl alcohol.

Cutting DNA

Explain how enzymes cut DNA

-Enzymes that cut DNA are called restriction enzymes and they are mostly found in bacteria. These enzymes are sequence specific, which means that they only bind and cut specific DNA sequences. After the restriction enzymes bind to a specifically recognized sequence, they cut down the sugar phosphate backbones of the DNA strands.

Find and compare 3 different enzymes (one which must cut with ‘sticky ends’ and another with ‘blunt ends’)

-AluI (source: Arthrobacter luteus) produces blunt ends

-BamHI (source: Bacillus amyloliquefaciens) produces sticky ends

-EcoRI (source: Escherichia coli) produces sticky ends

-AluI, which produces blunt ends, cuts the strand symmetrically whereas BamHI and EcoRI, producing sticky ends, cut the strand asymmetrically. This means that BamHI and EcoRI create 5’ or 3’ overhangs on the DNA strand, which makes it easy to ligate. On the other hand, enzymes such as AluI leave no overhang because it cuts the strand symmetrically.

Separating nucleic acids/protein

Outline and explain steps: purpose of gel, electricity, ‘running buffer’, DNA standard, ‘loading buffer’, ethydium bromide

-The purpose of gel is to sort out DNA strand by filtering them with its sponge-like consistency.

-The electricity during electrophoresis makes the DNA strands in the wells of the gel move through the gel via a current. The smaller the strand, the further it will go in the gel.

-Running buffer is mixed with agarose when making the gel so that electrical charges can flow through the gel

-DNA standard contains DNA strands with known lengths; therefore it is used as a reference to compare the strands from the DNA sample with those from the standard.

-A loading buffer contains a dye that makes the DNA sample visible as it goes through the gel. Furthermore it makes the DNA sample thicker so that it will stay in the gel well instead of floating away.

-Ethydium bromide is used to stain the DNA sample because it binds to the DNA and is visible under fluorescent light.

Amplifying DNA

Outline and explain steps: role of primers, heating, cooling, reheating, over and over, compare amount of DNA at start to amount of DNA at finish.

-The primers in PCR attach to sites on the DNA strand that are at either end of the segment that is targeted to be copied.

-Heating the DNA causes it to unwind into two separate strands. Cooling it then leads to the primers in the solution to attach to the targets before the two DNA strands can rejoin. The solution gets heated again, causing the DNA polymerase to attach on a primer and add complementary nucleotides onto the strand. The temperature is then heated, cooled, and heated again to repeat this cycle, creating more complementary strands of the target sequence. After 4 cycles, there are 8 fragments that contain only the target sequence. After 30 cycles, there are over a billion fragments that contain only the target sequence and 60 copies of the longer length molecules.