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Relationships and Biodiversity State Lab (Pg 131-146; you turn in 141-146) Relationships and Biodiversity State Lab (Pg 131-147) Plants No seeds seeds Gymnosperms Angiosperms (naked seed; pine, spruce, fir, etc…) (vessel seed (protective chamber); flowering plants) Relationships and Biodiversity State Lab (Pg 131-147) Relationships and Biodiversity State Lab (Pg 131-147) dicots monocots Angiosperms diverged into monocots and dicots Relationships and Biodiversity State Lab Angiosperms: Monocots vs. Dicots Relationships and Biodiversity State Lab PLANT VASCULAR TISSUE - tissues that conduct food and water (phloem and xylem) Relationships and Biodiversity State Lab 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. Relationships and Biodiversity State Lab Curol Curol prevents polymerization of microtubules, which would kill cancer cell because… Relationships and Biodiversity State Lab Microtubles (in yellow) are critical in cell division. Relationships and Biodiversity State Lab Curol Problem: Botanus curus is an endangered species and we cannot synthesize curol in a lab yet. What do we do? Botanus curus Relationships and Biodiversity State Lab 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? Relationships and Biodiversity State Lab 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: 1. Gather structural and molecular evidence to determine which plant species is most closely related to Botanus curus and therefore may also produce Curol. Relationships and Biodiversity State Lab 1. You will be separated into 7 groups of 3 or 4. 2. Each group will begin at one of the 7 stations. 3. There will be a rotation every 7 minutes. Relationships and Biodiversity State Lab 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. Relationships and Biodiversity State Lab 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. Relationships and Biodiversity State Lab Remember Paper chromatography? What famous individual used this technique and for what did Chargaff, the A,T,C,G ratios of DNA from this person use it to observe? Erwin different organisms - Analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. Relationships and Biodiversity State Lab 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. Relationships and Biodiversity State Lab Station 4 - Paper Chromatography to Separate Plant 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 pigments. 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… Relationships and Biodiversity State Lab Station 4 - Paper Chromatography to Separate Plant Pigments 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). 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). Relationships and Biodiversity State Lab Station 4 - Paper Chromatography to Separate Plant Pigments 3. Add about 1cm of water to each beaker. 4. Place two drops of plant extract from Botanus curus just above the pencil line as shown in Figure 2. 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. Pigment Sample MUST BE above the water level or the pigment will just diffuse into the water!!!!!! Relationships and Biodiversity State Lab Station 4 - Paper Chromatography to Separate Plant Pigments DO NOT contaminate the eye droppers!! Label with your name and staple the four pieces of chromatography paper to the lab when you turn it in. GOGGLES Relationships and Biodiversity State Lab Station 5 - Indicator Tests for Enzyme M Enzyme M is an enzyme in Botanus curus that is part of the Curol biosynthetic pathway. Relationships and Biodiversity State Lab 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. Relationships and Biodiversity State Lab Station 5 - Indicator Tests for Enzyme M - 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 eye droppers!! GOGGLES Relationships and Biodiversity State Lab Station 6 – Restriction fragment analysis using Gel Electrophoresis 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. Relationships and Biodiversity State Lab Station 6 – Restriction fragment analysis using Gel Electrophoresis 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 2. 3. Attach fragments to the lab when you turn it in. Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? 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… Aside: Why do these enzymes not cut the bacterial chromosome? The bacterial chromosome is methylated (modified by adding – CH3 groups so the enzymes can’t bind to it) Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? 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… Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Restriction enzymes Ex. EcoRI Notice anything interesting about this sequence? - It is palindromic, read the same way forward and backward. - Majority of restriction sites are palindromic… Fig. 12.4 Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Restriction enzymes Ex. EcoRI EcoRI 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 Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Restriction enzymes restriction enzymes Chapter 12 - DNA Technology and the Human Genome AIM: What are some other tools of DNA technology? Gel Electrophoresis This technique allows one to not only indirectly view the DNA, but also to separate and view the DNA fragments. Fig. 12.10 Chapter 12 - DNA Technology and the Human Genome AIM: What are some other tools of DNA technology? Gel Electrophoresis Gel (like jell-o) The gel is made of either agarose or polyacrylamide. It has tiny, microscopic pores that DNA can fit through. Fig. 12.10 Chapter 12 - DNA Technology and the Human Genome AIM: What are some other tools of DNA technology? Gel Electrophoresis Gel (like jell-o) The DNA sample is loaded in the wells at the top of the gel. One sample per well. Fig. 12.10 Chapter 12 - DNA Technology and the Human Genome AIM: What are some other tools of DNA technology? Gel Electrophoresis 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 gel?because the phosphates are negative. The DNAofis the negative Fig. 12.10 negative electrons move down and push (repel) the DNA down Chapter 12 - DNA Technology and the Human Genome AIM: What are some other tools of DNA technology? Gel Electrophoresis 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. Chapter 12 - DNA Technology and the Human Genome AIM: What are some other tools of DNA technology? Gel Electrophoresis 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… Fig. 12.10 Relationships and Biodiversity State Lab 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? Chapter 13 – How Populations Evolve AIM: What evidence do we have for evolution? 8 Number of different amino acids in hemoglobin. 125 The more recent the common ancestor, the more similar the amino acid sequence should be since the DNA should be more Relationships and Biodiversity State Lab 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. The sequence shown in the lab that you need to transcribe is the noncoding/anti-sense/template strand.