Relationships and Biodiversity. Relationships and Biodiversity. Relationships and Biodiversity State Lab (Pg )

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1 6/19/11 (Pg ) Plants No seeds (Pg ; you turn in ) seeds Gymnosperms (naked seed; pine, spruce, fir, etc ) (Pg ) Angiosperms (vessel seed (protective chamber); flowering plants) (Pg ) dicots monocots Angiosperms diverged into monocots and dicots Angiosperms: Monocots vs. Dicots PLANT VASCULAR TISSUE - tissues that conduct food and water (phloem and xylem) 1

2 Curol Curol Curol is a mitotic inhibitor used in cancer chemotherapy. It was discovered in a National Cancer Institute program at the Research Triangle Institute in 1967 when Monroe E. Curoli and Mansukh C. Cancurcere isolated it from the leaves of the tree Botanus curus. Curol prevents polymerization of microtubules, which would kill cancer cell because Microtubles (in yellow) are critical in cell division. Curol Problem: Botanus curus is an endangered species and we cannot synthesize curol in a lab yet. What do we do? Botanus curus Are there other species related to Botanus curus that also produce Curol? Scientists have found three candidates: Species X Botanus dienerhlus Species Y Botanus lolevinus Species Z Botanus liuginus Your goal is to gather evidence to try and determine which species might also produce Curol. What evidence might you gather? Are there other species related to Botanus curus that also produce Curol? Your goal: Scientists have found three candidates: Species X Botanus Dienerhlus Species Y Botanus Lolevinus Species Z Botanus Liuginus 1. Gather structural and molecular evidence to determine which plant species is most closely related to Botanus curus and therefore may also produce Curol. 2

3 1. You will be separated into 7 groups of 2 or Each group will begin at one of the 7 stations. 3. There will be a rotation every 7 minutes. Stations 1 and 2 - Structural Evidence Station 1 - Structural Characteristics of plant Compare the structural characteristics of the plant samples. Record your observations in Table 1. Station 2 - Structural Characteristics of Seeds Compare the structural characteristics of the seed samples. Record your observations in Table 1. Station 3 - Microscope Internal Structure of Stems 1. There are four microscopes. 2. Each microscope has a cross section of one of the species (Botanus curus, species X, Y, and Z). 3. Use the low magnification. Compare the arrangement of the bundles (circular or scattered) and record you observations using words or diagrams in Table 1. Remember Paper chromatography? What famous individual used this technique and for what did Erwin Chargaff, the A,T,C,G ratios of DNA from this person use it to observe? different organisms - Analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. - The leaves of Botanus curus, species X, species Y and species Z have been crushed up for you and an ethanol extraction was performed to pull out the partially hydrophobic pigments. How does Paper chromatography work? The solvent (water in this case) will wick (travel) up the paper. The solvent will eventually make its way to the sample absorbed on the paper. The molecules in the sample will have a different affinity for both the paper and the solvent. If the affinity is high for the solvent, the molecule will move quickly up the paper with the solvent. If the molecule has a high affinity for the paper, it will resist movement and only move slowly up the paper. What should you do with the pigment extract? You should run them each on paper chromatography to separate the pigments and compare the different species. The species most similar to B. curus would be expected to have similar pigment molecules since pigments are made by enzymatic reactions and therefore the species would have similar enzymes 3

4 6/19/11 1. Take four strips of chromatography paper and try to straighten them the best you can by curling them in the opposite direction and putting a slight crease down the center (see example setup on the bench). 3. Add about 1cm of water to each beaker. 2. Draw a line 2 cm from the bottom of each of the four chromatography papers. Use a pencil to label the top edge of the chromatography paper either Bc (Botanus curus), X, Y, or Z (look at Figure 2 in the lab manual). 5. Place the paper into the water and allow the water to move up the paper. Repeat steps 4 and 5 for the other samples. 4. Place two drops of plant extract from Botanus curus just above the pencil line as shown in Figure 2. Pigment Sample MUST BE above the water level or the pigment will just diffuse into the water!!!!!! Station 5 - Indicator Tests for Enzyme M DO NOT contaminate the samples!! Enzyme M is an enzyme in Botanus curus that is part of the Curol biosynthetic pathway. Label with your name and staple the four pieces of chromatography paper to the lab when you turn it in. GOGGLES Station 5 - Indicator Tests for Enzyme M Station 5 - Indicator Tests for Enzyme M - It is not possible to test a plant directly for Curol (concentration is too low). - However, if enzyme M, an enzyme known to be a part of the Curol synthesis pathway, is present, the plant may produce Curol. - Test the plant extract of each species for enzyme M by adding 5 drops of each extract to the substrate for enzyme M. The product of enzyme M is a non-hazardous gas and therefore bubbling indicates that enzyme M is present. - Record the results in Table 1. DO NOT contaminate the samples!! GOGGLES 4

5 Station 6 Restriction fragment analysis using Use restriction fragment length analysis to determine relatedness between the different species. The species that is more closely related to B. curus should have more similar restriction sites in its DNA and therefore will produce similar length fragments when run on a gel. Station 6 Restriction fragment analysis using A. Use the colored DNA sequence strips, which represent the DNA of each species B. Do a restriction fragment analysis using a restriction enzyme (scissors) that cut at CCGG. 1. Cut all CCGG sites 2. Run a simulated gel electrophoresis using Table 2. Draw a line indicating the size of the band on Table Attach fragments to the lab when you turn it in. Recall how a bacterium defends itself when a bacteriophage injects its DNA into a bacterium The bacterium has enzymes called restriction enzymes that attempt to cut up the bacteriophage DNA before it can take over the cell. Different species have different restriction enzymes 1. molecular DNA scissors (enzymes that cut DNA) 2. Different restriction enzymes cut different sequences. 3. Scientists have isolated hundreds of different restriction enzymes from many different bacteria EcoRI, BamHI, NcoI, etc Aside: Why do these enzymes not cut the bacterial chromosome? The bacterial chromosome is methylated (modified by adding CH 3 groups so the enzymes can t bind to it) Ex. EcoRI Ex. EcoRI EcoRI Notice anything interesting about this sequence? - It is palindromic, read the same way forward and backward. - Majority of restriction sites are palindromic Fig Notice that is doesn t cut straight through like paper scissors. The enzyme cuts each strand after the G nucleotide generating single-strand regions called sticky ends. 5

6 restriction enzymes This technique allows one to not only indirectly view the DNA, but also to separate and view the DNA fragments. Gel (like jell-o) Gel (like jell-o) The gel is made of either agarose or polyacrylamide. It has tiny, microscopic pores that DNA can fit through. The DNA sample is loaded in the wells at the top of the gel. One sample per well. Electricity (electrons flow from top of gel by the samples to the bottom of the gel) Electricity is then run through the gel. Why do you think the negative end is on the sample side and the positive end is on the other end of the gel? DNA is negative because the phosphates are negative. The negative electrons move down and push (repel) the DNA down with them. Which will move faster through the micro-porous gel, the longer DNA fragments or the shorter DNA fragments? The small fragments (fewer nucleotides) will move more easily through the gel and hence go faster than the large ones. 6

7 Station 7 Transcribe and Translate the DNA of each organism to determine if an enzyme in species X, Y and Z has a similar amino acid sequence indicating a close relationship. What can you say about the amino acid sequence of the same protein in two different species if they have a recent common ancestor? The gel is soaked with a a compound called ethidium bromide, which sticks to DNA and lights up when you hit the gel with UV light Chapter 13 How Populations Evolve AIM: What evidence do we have for evolution? 8 Station 7 Transcribe and Translate the DNA of each organism to determine if an enzyme in species X, Y and Z has a similar amino acid sequence indicating a close relationship. Number of different amino acids in hemoglobin. 125 The sequence shown in the lab that you need to transcribe is the noncoding/anti-sense/template strand. The more recent the common ancestor, the more similar the amino acid sequence should be since the DNA should be more similar. 7