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Tested Studies for Laboratory Teaching
Volume 18

Boston University, June 11-15, 1996

[Out of print - available in full text as PDF files]
Editor: Jon C. Glase (Cornell University)
Host: Elizabeth Godrick

Appendix A: Abstracts of Mini Workshops  [full text]

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Abstracts (Vol. 18)

Laboratory Exercises in Cell and Molecular Biology, Genetics, and Development

1 -- Use PCR and a Single Hair to Produce a "DNA Fingerprint"
A. Malcolm Campbell, John H. Williamson, and Diane Padula  [full text]
Key words: PCR, Fingerprint, VNTR, D1S80, APOC2, Forensics, Hair
As a former student once commented, "PCR is the sliced bread of molecular biology". Polymerase Chain Reaction (PCR) is a process that allows an investigator to amplify a portion of a single piece of DNA into over one billion copies. We have used PCR with our Introductory Biology students to generate a simple "DNA fingerprint". This exercise will outline all the steps required to generate a DNA fingerprint for each student during two lab meetings. This article covers the background information concerning the relevant techniques and the specific details required for students to isolate their own DNA from a single hair follicle, prepare their DNA for PCR, load an agarose gel, and analyze their DNA samples.

2 -- Lighting Up: Analysis Of Yeast RAS Genes Using A Chemiluminescent Southern Blot Procedure
Karen A. Malatesta and Linda B. Cholewiak  [full text]
Key Words: Southern, blot, chemiluminescence, transfer, electrophoresis, yeast, RAS
Genomic and plasmid-encoded RAS2 genes from Baker's and brewing yeast strains will be analyzed using a shortened Southern blot procedure employing chemiluminescence. Unique parts of this procedure are the gravity-driven "upside down" transfer and a rotisserie-like hybridization oven, which greatly facilitate shortening the transfer time to 45 minutes and the hybridization step to 60 minutes. Chemiluminescence obviates problems associated with radioisotopes without sacrificing sensitivity: the plasmid-borne RAS gene can be visualized within 15 seconds and the single-copy yeast genomic RAS2 gene, within a minute. Topics for discussion related to this laboratory can include the concepts of RFLPs, evolutionary conservation of DNA sequences and the genetic basis of cancer.

3 -- Purification of Maltose-Binding Protein from E. coli Periplasm
Judith R. Levin  [full text]
Key Words: protein purification, affinity chromatography, protein determination, standard curve, SDS-PAGE, cell fractionation, osmotic shock
By correlating the protein concentration of various fractions from E. coli with the appearance of samples on a gel, this exercise introduces beginning students to the quantitative "bookkeeping" involved in protein purification. A periplasmic fraction is isolated by osmotic shock, and affinity chromatography with amylose resin is used to purify maltose-binding protein. Visualization of fractions by SDS-polyacrylamide gel electrophoresis allows students to evaluate the qualitative success of the purification and to estimate the subunit molecular weight of the purified protein.

4 -- Cell Fractionation in Tetrahymena
Nancy Rosenbaum  [full text]
Key Words: ell fractionation, nucleus, Tetrahymena thermophila, hemacytometer
To illustrate cell fractionation, nuclei are isolated from the ciliated protozoan, Tetrahymena thermophila. A table top clinical centrifuge is used for the fractionation steps and the procedure is monitored microscopically using a differential stain. To determine the efficiency of the procedure, cell and nuclear counts are determined with a hemacytometer. To quantify DNA, the Diphenylamine Reaction is carried out and the amount of DNA per nucleus is calculated.

5 -- Isolation of Myosin and Actin from Chicken Muscle
Richard H. Racusen and Katerina V. Thompson   [full text]
Key Words: Muscle, protein electrophoresis, myosin, actin, biochemistry
Using modifications of classic biochemical procedures for purifying the muscle contractile proteins, myosin and actin, we devised a 3-hour laboratory on the biochemistry of muscle that is appropriate for students in an introductory college biology course. The protocol begins with the extraction of small pieces of frozen chicken muscle in high salt (enhances myosin recovery) and low salt (enhances actin recovery) buffers. Solubilized myosin is induced to form visible filaments by lowering the ionic strength of the buffer, and a small pellet of enriched myosin is collected by centrifugation. Aliquots of both myosin and actin preparations are prepared for electrophoresis on small SDS, polyacrylamide slab gels. Completed electropherograms are stained, and students determine the molecular weights of gel bands. These data are then compared to known structural models of myosin and actin subunits and fragments.

6 -- Identification of T Lymphocytes
James M. Bader   [full text]
Key Words: immunization, lymphocyte, cell-mediated immunity, rosette.
In a clinical setting, it is often necessary to examine the cells of the immune system, especially in patients with immune deficiencies. One method takes advantage of the fact that lymphocytes will bind foreign red blood cells to form rosettes which can be enumerated microscopically. Specifically, the Ox49/50 antigen on the rat T lymphocyte surface has been
identified as the probable binding site of the sheep rbcs. In this exercise, we will show that the number of antigen-binding lymphocytes increases after immunization.

Laboratory Exercises in Physiology

7 -- Water and Solute Movement Through Red Blood Cell Membranes
Stephen Hunt  [full text]
Key Words: N2 Fixation, nitrogenase, H2 evolution, nodule, soybean, gas exchange, oxygen, regulation.
Measurement of N2 fixation is rarely conducted in undergraduate teaching laboratories because traditional methods are both hazardous and expensive. In the following exercise, measurement of H2 evolution from nodulated legume roots provides a safe, non-invasive method to assay nitrogenase activity in vivo. The method also allows students to investigate aspects of the nitrogenase mechanism, and to study effects of environmental changes  on N2 fixation. Specifically, students measure the relative allocation of electron flux through nitrogenase to protons and N2, and then investigate effects of O2 supply and nodule detachment on nitrogenase activity.

8 -- Pigment Granule Transport in Chromatophores
Sally E. Nyquist and Kathryn B. Toner  [full text]
Key Words: chromatophores, melanophores, intracellular motility, microtubules, second messengers, hormonal regulation, neural regulation.
This laboratory offers students the opportunity to investigate factors regulating migration of pigment granules within the cytoplasmic extensions of fish scale melanophores, a process which results in darkening or lightening of the fish. The fish scale is removed, used intact or permeabilized, and viewed via light microscopy to detect aggregation/dispersion. Using this system, pairs of students design and execute experiments testing the effect of potential regulatory factors (neural and hormonal) on dispersion/aggregation of pigment granules. Intracellular second messenger compounds may also be investigated. The importance of controls and critical analysis of experimental results are emphasized.

9 -- Preparation of Mammalian Meiotic Chromosomes and Spermatozoa/Obtaining Early Mammalian Embryos and Preovulation Oocytes
Frank J. Dye  [full text]
Key Words: chromosomes, embryos, mammals, meiosis, mice, mitosis oogenesis, ovaries, oviducts, spermatogenesis.
The objectives of this workshop are to: introduce participants to a method for obtaining spreads of chromosomes from mammalian testes (which will include both mitotic and meiotic chromosomes), reinforce the participants' knowledge of meiosis and spermatogenesis by critical interpretation of what is seen through the microscope, obtain oocytes from mouse ovaries, obtain living embryos from oviducts of the mouse, and note the dynamics of the oviduct, including smooth muscle contraction of the oviduct and movement of embryos within the lumen of the living oviduct.

10 -- Olfaction and Chemical Communication
James Traniello  [full text]
Key Words: olfactory communication, pheromones, humans, ants, termites.
This laboratory examines olfactory communication in humans, ants, and termites. The study of the relationship between molecular structure and odor perception in insects and human response to different odorants is followed by an experimental analysis of chemical signaling in ants and termites. These latter exercises demonstrate the role of the physical properties of chemicals in pheromone communication and the measurement of factors important to the analysis of the biological properties of pheromones.

11 -- Integrating Introductory Biology and Chemistry Laboratories: Human Metabolism of Vitamin C and Fruit Juice Analysis-an Example
Patricia Samuel and Elizabeth Godrick  [full text]
Key Words: Vitamin C, integrated biology/chemistry, human, fruit juices.
Using the study of Vitamin C as an example, this workshop addresses a method by which first year biology and chemistry laboratory sections can be integrated. The modular laboratory program is presented from the perspective of biologists and chemists. Emphasis is placed on the design of human studies and attention to titration accuracy. Human metabolism of Vitamin C and analysis of Vitamin C content of fruit juices will be quantitated.

Laboratory Exercises in Evolution, Ecology, and Behavior

12 -- Orientation of Marine Invertebrates to Odor Sources
Rainer Voigt and Jelle Atema  [full text]
Key Words: odor dispersal, flow visualization, chemoreception, orientation behavior, marine invertebrates.
This workshop demonstrates several techniques to visualize flow patterns in the aquatic (marine) environment. Flow is essential not only for breathing and excretion but also for sending and receiving chemical signals for communication. The dispersal pattern of odors depends on the water currents in the environment and odor release patterns under the animal's control. The latter include animal-generated "information currents". We demonstrate how odors affect the sampling strategies of marine invertebrates and how they control their fluid environment. In addition, we demonstrate how marine invertebrates orient toward an odor source in a straight flume or in a Y-maze.

13 -- Phenotypic Variation in Plants
Lawrence Blumer  [full text]
Key Words: phenotype, genotype, environment, ecotypes, heritable variation, anthocyanin, Brassica rapa.
Phenotypic variation in any natural population results from a combination of environmental and genotypic variation. The causes for variation in one species will be evaluated by manipulating environmental conditions for three varieties of rapidly growing mustard plant, Brassica rapa . Physical and biochemical traits of plants grown under different conditions can be quantified to determine the causes of observed trait variation.

14 -- Herbivory and Anti-herbivory: Investigating the Relationship Between the Toxicity of Plant Chemical Extracts and Insect Damage to the Leaves
Kathy Winnett-Murray, Lori Hertel, and K. Greg Murray  [full text]
Key Words: allelochemics, bioassay, brine shrimp, plant defenses, herbivory, leaf damage, toxicity, insect herbivores, defense compounds.
Both field and laboratory components are used to explore some consequences of the chemical arms race between plants and insect herbivores in this open-ended investigation. Students will quantify herbivore damage on different individuals of a selected tree species (e.g. Red Oak, Quercus rubra). This will form the basis for individual hypotheses to determine the relation between leaf damage and the production of defensive toxins in the same individuals. Students will prepare crude chemical extracts from leaves collected from individual plants sustaining varying degrees of insect damage. The toxicity of these extracts is evaluated in a bioassay based upon brine shrimp survival over a 24 hour period.

15 -- The Intertidal Environment Of The Rocky Coast
Elaine Senechal-Brown and Harlan K. Dean  [full text]
Key Words: competition, ecology, field study, intertidal, marine, predation, rocky coast, zonation.
Rocky shores predominate the Atlantic coast of the U.S. and Canada north of Cape Cod. The ruggedly sculptured rock faces have been weathered by waves and sea-level changes over the ages. The intertidal of this coast is a harsh environment, alternately exposed and submerged with each tide, pounded with the force of the waves; yet here is found a rich diversity of marine life. The organisms of the rocky intertidal have a unique set of adaptations which allow them to tolerate a wide variety of environmental conditions and there is great competition for the limited space of the rock surfaces. This zonation is a characteristic feature of the rocky intertidal and is the result of complex interactions of both physical and biological factors. Because of its accessibility from land at low tide the rocky intertidal is uniquely suited for students to investigate the ecology of its marine life. In this workshop we will explore the rocky intertidal coast and introduce several field studies that can be used in this environment.

16 -- Avian Niche Partitioning
Fred Wasserman  [full text]
Key Words: bird behavior, field study, niche, competition.
The purpose of this laboratory is to help students examine differences in competitive ability among avian species at bird-feeders. Differences in methods to obtain resources and in ability to fight and to exclude other species are observed. The laboratory is designed to run on either an urban or rural campus. In Boston we take public transportation to the field site and return within a three hour laboratory period.

Mini Workshop

 [full text]

• Bioinformatics: The Retrieval and Analysis of DNA and Protein Sequences (Tongjia Yin and Janice Lovett)
• Human Nutrition: Using a Computer Program to Determine How Well You Eat (Wayne L. Silver)
• Teaching Evolutionary Pathways with Imaginary Animals (Richard W. Miller)
• A Multimedia-based Lab Manual: To Enrich, Improve and Expand Learning and Teaching in the Wet Lab (Gabriela Wienhausen)
• How to Get Students to Stop Asking, "Is This Lab Going to Take the Whole Three Hours?: A Description of a Cell Biology Lab Curriculum Which Emphasizes Scientific Inquiry (Donna M. Bozzone)
• Selected Laboratory Experiments for Elementary Education Major or Other Non-Biology Majors (Denise A. Martin)
• A Qualitative and Quantitative Approach to Investigating Cellular DNA Repair Mechanisms (Glenn A. Bauer)
• Why Should I Wear Sunscreen? Ultraviolet Radiation, DNA, And Mutations (Sheila Kratzer and Charlene M. Waggoner)
• Using the Scientific Method to Study Optical Orientation in Blowfly Larvae (William D. Blaker)
• Non-Majors Environmental Biology: Large Class Hands-On Experiences (Ruth E. Beattie)
• Lights, Bean Plants, Action: Starch Printing, a Student Exercise (Carol Reiss)
• DNA: From Lab to Courtroom (Catherine A. Teare Ketter)
• Species Identification and Systematics (Linda T. Collins and Charles H. Nelson)