A ribosome is composed of a large subunit and a small subunit and each subunit contains specific RNAs that are associated with ribosomal proteins. Ribosomes play the same essential role in protein synthesis in all contemporary organisms and the genes for ribosomal RNAs in each subunit are highly conserved. The gene for the small-subunit ribosomal RNA (16 S-like) has been especially useful in evolutionary studies of distant phylogenetic relationships because it has remained quite stable during evolution of all organisms.
In this experiment, students prepare a protein extract from cow heart. They then determine the molecular weights of major contractile proteins by comparing their migration on SDS-polyacrylamide gels to the migration of standard proteins of known size. Students also identify and determine the molecular weight of the major proteins found in milk. This exercise requires 1 three-hour or 2 two-hour lab sessions and a table top centrifuge or microcentrifuge is required. Typical results of the exercise are shown on the gel.
Satellite DNAs are highly repeated sequences of unknown function. The satellite DNA from the meal worm beetle is remarkable since it represents over 50% of this insects genome. In this exercise, students first isolate DNA from beetle larvae by a simple and safe procedure. They then digest the DNA with EcoR1 and examine the satellite DNA following electrophoresis as shown on the gel pictured below. Suffient materials are provided so that the experiment can be carried out twice by eight groups of students.
This miniprogram was designed to give students a basic understanding of enzyme kinetics. In the first experiment in this series, students prepare an extract from wheat germ. They then determined the initial velocity (Vo) of the reaction catalyzed by purified acid phosphatase and by the acid phosphatase activity present in the extract. From these data, they estimate the amount of the enzyme that is present in the wheat germ. In the second experiment, the student examines the effects of substrate concentration on the reaction velocity.
Genes introduced into E.coli by plasmid-mediated transformation can confer variation in the phenotype of the bacteria. For example, E.coli containing the ampicillin-resistance gene grows in the presence of this antibiotic while the product of the ß-galactosidase gene enables the bacteria to convert the ß-galactosidase substrate X-gal to a blue product. Similarly, E.coli containing the Lux operon produce colonies that glow in the dark.
A limitation to the Southern blotting procedure for the teaching laboratory is that it requires multiple laboratory sessions. The procedures have been streamlined for this experiment so that they can be carried out in a single 3-hour laboratory period. In the exercise, students electrophorese three unknown DNA samples on an agarose gel. One of the samples (instructor keyed) contains biotinylated DNA from the bacterial virus lambda.
Each protein carries in its amino acid sequence information pertaining to its evolutionary history and origin, and provides clues to the evolutionary history of the organism in which it is found. Indeed, proteins existing today are in effect living fossils. This concept is illustrated in this exercise where eight groups of students examine the abilities of antibodies against cow gamma globulin to react with gamma globulins in the sera of cow, goat, sheep, horse, and chicken.
IND-18. An Introduction to Zoology
Molecular and cell biology can be understood and appreciated only in the light of their relations to the whole organism. However, in recent years it has become fashionable to reduce or to eliminate altogether laboratory time allotted for organismic biology. In this program, students prepare and then study cross sections of planaria, earthworm, lamprey (larvae), and tadpole. The laboratory guides emphasize hierarchies of animal complexity and the similarities and differences between the invertebrates and vertebrates.
Students prepare and then study sections of trachea and esophagus in order to identify and characterize the major tissue types ( epithelia, connective tissue and muscle). They then study selected organs including ovary, testis, intestine, spinal cord and skin.
Locating specific proteins and nucleic acid molecules in tissue sections is an important goal in cell biology. An effective and simple technique for this purpose is tissue printing which permits the localization of specific macromolecules in animal and plant tissues. Here students perform this technique to examine the tissue distribution of the enzyme peroxidase in plants. First, students section carrots, celery, and other vegetables with razor blades and transfer the proteins from the cut sections to nitrocellulose membranes by application of gentle pressure.
Purified DNA is most often used as a template in the PCR reaction. However, it is possible to amplify specific DNA sequences without DNA purification by starting with a single living E. coli colony. This technique is known as colony PCR and provides a powerful and reliable method for the rapid amplification and isolation of any gene in the E.coli genome or any gene on a plasmid that is carried by E.coli. In this exercise, students carry out colony PCR starting with a culture of E. coli that carries an ampicillin-resistance gene on plasmid pUC18.
This program was designed to actively engage students in exciting biological research projects of their own design. The projects focus on peroxidases, which form a large family of related enzymes that are ubiquitous in plants. Plant peroxidase isoenzymes can be tissue specific, developmentally regulated and display variable tissue and, high salt and disease resistance defense reactions and this induction may be related to the abilities of peroxidases to strengthen the plant cell wall and to kill microorganisms.
This economical 2-part exercise provides an introduction to agarose gel electrophoresis. In the first part of the exercise, students identify unknown dye molecules by comparing their electrophoretic migration with the migration of known dyes. In the second part students identify dye molecules that bind to DNA and determine the mechanism. The exercise was designed for 8 groups of students and includes four colorful dye mixtures and DNA.
Electrophoresis Package 1/8 is needed but not provided.
The five histone molecules are the major proteins that associate with DNA to form nucleosomes. In this experiment, students isolate nuclei from chicken erythrocytes and then prepare nucleosomes from the erythrocytes by using a simple procedure that employs micrococcal nuclease. They then determine the molecular weights of the five histone proteins by comparing their migration on SDS-polyacrylamide gels to the migration of standard proteins of known size. This exercise requires a single two-three hour lab session and a microcentrifuge is required.
Microtubules are hollow cylinders made up of polymers of the protein tubulin. Microtubules are major components of cilia and flagella, which are tail like projections that are covered by a plasma membrane and extend outwards from the cell. Motile cilia are used for locomotion and food gathering by some protozoa and are found in the lining of the trachea, where their wave like motion propels mucus, dust and other foreign matter out of the lungs.
The primary level of chromosome structure in eukaryotes occurs when the DNA molecule is wrapped around histone proteins into particles called nucleosomes. Evidence for this “beads on a string” model is derived from nuclease digestion studies. When nuclei are incubated with micrococcal nuclease, the enzyme cleaves the linker DNA between nucleosomes (the string) but not the nucleosomal core DNA (the beads).
Synthetic biology is an exciting new field that uses engineering principles and mathematical modeling to design and construct biological devices. Synthetic biology projects include the construction of bacterial computers that can solve mathematical problems. Microbial machines, in the form of genetically engineered E. coli cells, have solved a variety of mathematical problems, which have had important applications in biology, medicine and technology.
IND-29 Electrophoretic and Chromatographic Analysis of Photosynthetic Pigments from Blue-Green Algae. (View Individual Experiment)
Cyanobactera, also known as blue-green algae, obtain their energy by photosynthesis using sunlight as their energy source. These organisms have been considered to be the oldest and the most important bacteria on the earth. It is believed that they were responsible for the initial oxygenation of the earth's atmosphere through photosynthesis and it is also felt they were the precursors to the chloroplasts that are found in true algae and plants. There are two classes of photosynthetic pigments in Cyanobactera.
The ELISA (enzyme-linked immunosorbant assay) is a powerful immunological method for detecting specific proteins in complex protein mixtures. The ELISA has become an important tool for the cell and molecular biologist. It is increasingly being applied in clinical medicine for detecting proteins associated with disease including antibodies produced in response to infection by the HIV virus. The method is also easy to perform and yields graphic results making it well suited for the teaching laboratory.
There are three experiments that make up this program.
IN THE FIRST EXPERIMENT, students are given the opportunity to determine the amount of amylase in their own saliva and in cell-free extracts prepared from the cow pancreas. This exercise offers practical experience with a novel enzyme assay and introduces the concept of the calibration curve for determining the levels of a specific enzyme in a complex mixture.
In most mammals, antibody production does not occur until after birth. The newborn calf receives antibodies in milk and these antibodies are responsible for passive immunity during early postnatal life. Thus, fetal calf serum is devoid of antibodies, neonatal calf serum contains antibodies of maternal origin while serum from the adult contains large amounts of antibodies made by the mature immune system. This fascinating developmental scheme is illustrated in this exercise.
The binding of an enzyme to its substrate is only one example of the many specific molecular interactions that occur in biological systems. An analogous binding process occurs with serum albumin which binds certain small molecular weight compounds and serves as a carrier molecule for these compounds in blood. In this exercise, students use an electrophoretic assay to examine the binding of various dyes to albumin. The results of this graphic analysis show that the binding of dyes to albumin is saturable, specific, compatible, and dependent on the native structure of the protein.
A mutation is a change in the nucleotide sequence of DNA which leads to an inherited change in an organism. Restriction endonucleases provide valuable tools for characterizing mutations at the DNA level. This principle is illustrated in the exercise where students digest a normal and a mutant gene with EcoR1 and Hae III and then analyze the DNA fragments from each by electrophoresis as shown in the figure below. The gene is from rabbit and codes for the ß-globin chains of hemoglobin.
The polymerase chain reaction (PCR) is one of the most powerful techniques used in molecular biology . With this method, a few ng of DNA can be amplified millions of times in a test tube in a few hours. The PCR has been used extensively in studies of gene structure and function. The method is also becoming increasingly important in DNA typing procedures such as DNA fingerprinting and in the identification and characterization of mutations that cause human diseases. This exercise is designed to illustrate the PCR in the teaching laboratory.
The emission of light by living organisms is a fascinating process. The genetic system required for luminescence in the bacterium Photobacterium (Vibiro) fischeri is the lux operon. This operon contains a gene for luciferase (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). In this exercise, students create a luminescent population of bacteria by introducing into E.coli a plasmid that contains this lux operon.