from the National Science Foundation

PIs:

Frank J. DeToma

Craig T. Woodard

Department of Biological Sciences

Mount Holyoke College

South Hadley, Massachusetts 01075

Labs we've developed for Biological Sciences 210 (Genetics and Molecular Biology)

Labs we've developed for Biological Sciences 340 (Eukarotic Molecular Genetics)

INTEGRATION OF MOLECULAR BIOLOGY (AND COMMUNITY-BASED LEARNING) INTO THE BIOLOGY CURRICULUM
When we applied for this award, a priority for the Mount Holyoke College Department of Biological Sciences was to integrate the concepts, techniques, and applications of molecular biology into the entire biology curriculum. At the time, only a limited number of students had been able to gain experience in molecular biology - primarily through advanced biochemistry coursework or senior honors research. Our goal was to give undergraduates an appreciation of molecular biology and its methodology beginning in their first year of study. With the help of this grant, we have integrated molecular biology into the our department's curriculum at the introductory, intermediate, and advanced levels. We have constructed a molecular biology teaching lab, and assembled mobile molecular biology workstations, which can be used in laboratory courses and in independent student projects. The following are descriptions of some of the molecular biology projects that students have done in the teaching labs associated with our courses.

Biological Sciences 100 (Science of Life) (Introductory biology for non-majors)
We have made molecular biology an integral part of the lab experience in our introductory courses. We have introduced several molecular biology projects into the introductory laboratory biology lab, including:

-an experiment in which the students determine the difference between normal and mutant hemoglobin genes by restriction mapping.

-an experiment in which students perform DNA fingerprinting analysis using Polymerase Chain Reaction (PCR).

-an experiment in which students perform DNA fingerprinting analysis using Restriction digestion analysis.

Interdepartmental 121-222 (The Unity of Science)
In this course, which stresses connections between astronomy, biology, chemistry, geology, math, and physics, students learned molecular biology in lectures, discussions and by doing hands-on projects in the lab. The lab projects included:

-an experiment in which students transform bacterial cells with a recombinant plasmid containing the gene encoding Green Fluorescent Protein (GFP) and detect the transformed cells because they glow green when illuminated with ultraviolet light.

-an experiment in which students perform DNA fingerprinting analysis using Polymerase Chain Reaction (PCR).

Biological Sciences 210 (Genetics and Molecular Biology)
In the fall of 1997, we began offering a completely revised version of Biological Sciences 210, which was previously called "Genetics". In its new form, this course is entitled, "Genetics and Molecular Biology". The laboratory component of Genetics and Molecular provides most of our biology students with their first opportunities to perform in-depth analyses using molecular biology techniques. Students in Biology 210 have performed the following studies:

Screen for P-Transposable Elements in a wild-type population of Drosophila melanogaster
In this 7-week project, students used molecular techniques to screen for P-transposable elements (P-elements) in a strain of the fruit fly, Drosophila melanogaster, that was isolated on campus. The Blanchard Wild-Type strain is descended from a single female fly trapped in the Blanchard Campus Center cafeteria. The students did Southern blot hybridization analyses in attempt to determine whether or not Blanchard Wild-Type flies had P-elements in their genome. Student data indicated the presence of multiple P-elements in this strain.
Once the Biology 210 students determined that Blanchard Wild-Type flies had P-elements, they attempted to clone these P-elements by constructing genomic libraries from this strain. A plasmind vector was used in the construction of the library, and the students transformed E. coli cells with their libraries and plated them. The students did colony lifts onto nitrocellulose membranes, and hybridized the lifted libraries to detect colonies containing cloned P-element sequences. This was a combined group effort, and thousands of colonies were screened in this way. Twelve positively hybridizing colonies were detected in this way. The students isolated and restriction-mapped recombinant plasmids from these colonies.

Determining the sex of plants by Polymerase Chain Reaction (PCR)
In this project, students used a PCR-based strategy to determine the sex of individual plants of the species, Silene dioica. We grew plants in the greenhouse and also collected them in the field. Silene dioica is dioecious and has discernible X and Y chromosomes. In this study, students used the Random Amplified Polymorphic DNAs (RAPDs) genetic fingerprinting technique to find molecular markers for the Silene Y chromosome. DNA samples from known males and females (i.e., mature plants) were run on the same gel with unknown tissue samples. Students used the Y chromosome markers to identify your unknowns as males or females.

Biological Sciences 340 (Eukaryotic Molecular Genetics)
In the spring of 1997, we added Eukaryotic Molecular Genetics (Biol. 340) to our permanent list of course offerings. In this advanced course, we examine the role of molecular genetic analysis in the study of phenomena such as human disease (particularly breast cancer), animal development and programmed cell death. We also take a detailed look at the science behind the Human Genome Project. There are group discussions of original research articles and review articles, and we began using a case method approach this past semester. The laboratory component of Biological Sciences 340 now provides students with the opportunity to do investigative molecular biology. Several of the projects are closely-related to Mr. Woodard's own research (thus the frequent use of Drosophila), so the students are able to participate in a genuine research effort. At the beginning of the course, the students are provided with several ideas for good projects to do in the lab. They work in small groups, and the members of each group can decide which project that group will work on. If a student would prefer to try a project of your own design rather than one of the suggested projects, the instructor is happy to discuss that possibility with her. The following are some of the suggested projects that students have worked on.
These descriptions are directly from the course materials, and thus are directed to the students.

Suggested Project Mapping deletion mutations in Drosophila melanogaster
You will usually work in pairs, but sometimes in larger groups. Each pair will receive a number of different fruit fly stocks (a stock is a group of genetically-identical flies), including wild-type controls, and mutants. The mutant stocks carry deletion mutations resulting in abnormal development. Your job will be to map the breakpoints of these deletions.

Suggested Project: Cloning a gene by transposon-tagging
You will be given mutant fruit flies that have transposable elements called P-elements inserted in their genomes (a transposable element is a short DNA sequence that can, under certain conditions, move around the genome, and insert in different chromosomal locations). You can use a strategy taking advantage of polymerase chain reaction (PCR) to isolate genomic DNA adjacent to the inserted P-element, in an attempt to clone the mutated gene. The next step will be to screen a genomic library in an attempt to isolate the complete gene.

Suggested Project: Search for homologs of the Drosophila melanogaster E93 cell death gene in other species
The E93 gene of Drosophila melanogaster plays a key role in initiating programmed cell death in tissues fated to die during metamorphosis (the dramatic transformation from larva to adult). E93 was disovered relatively recently, and little is known about its mechanism of function. Discovery, isolation, and characterization of E93 homologs in other insect species should provide valuable insights into the function of this gene.

Goals of this project
1) determine whether or not various other insect species have genes homologous to E93.
2) Isolate E93 homologs from a number of these other insect species.
3) Sequence these E93 homologs.
4) Compare the sequences of the E93 genes from Drosophila melanogaster and the other species to try to learn about function.

Realistically, we can only hope to accomplish goal #1 this semester!

Suggested Project: Extension of the map of the FTZ-F1 gene of Drosophila melanogaster (*It should be noted that this exercise was carried out prior to the completion of the Drosophila Genome Project.)
Steroid hormones control gene expression, and a wide range of developmental processes in higher organisms, including humans. The biological processes controlled by steroid hormones include the development of secondary sex characteristics, reproductive function, and dietary metabolism. Metamorphosis in insects is directed by a single steroid hormone called ecdysone. By examining the mechanisms whereby ecdysone regulates metamorphosis in the fly, we hope to gain a better understanding of how steroid hormones control developmental processes in general. In order to determine how ecdysone controls the complex process of metamorphosis, we are studying the genes that are regulated by ecdysone during Drosophila metamorphosis. The FTZ-F1 gene encodes a nuclear hormone receptor superfamily member that appears to play a central role in directing stage-specific genetic and developmental responses triggered by ecdysone. Our work indicates that FTZ-F1 mediates the ecdysone-response of a set of other regulatory genes, enabling them to be induced by the hormone at the appropriate developmental times. In directing the precise timing of target gene induction by ecdysone, FTZ-F1 ensures that developmental events occur at the correct time, and in the correct temporal order.
In order to examine the role of ßFTZ-F1 in development, we are performing a molecular characterization of the gene. We have been performing a chromosome walk in an attempt to clone the entire FTZ-F1 gene. We have thus far characterized recombinant DNA molecules obtained in a screen of 2 different Drosophila genomic libraries, but we have as yet been unable to complete the map of FTZ-F1. It appears that the available genomic libraries are missing clones that would extend our map. This being the case, we
need to construct a new genomic library that will contain the clones we need. By constructing and screening this libray, we hope to complete the FTZ-F1 genomic map.

Goals of this Project
1)Construct a size-selected Drosophila melanogaster genomic library using a bacteriophage l vector.
2)Screen the library, using a DNA fragment from the extreme upstream end of the existing map.
3) Isolate and characterize positively-hybridizing clones.

Note: You may sometimes have to work briefly in the lab during times other than our normal lab meeting time!

INTEGRATION OF COMMUNITY-BASED LEARNING (AND INVESTIGATIVE LEARNING) INTO THE BIOLOGY CURRICULUM

Biological Sciences 327 (Microbiology)
The laboratory in this course made use of the centrifuges purchased using funds from this grant. The Microbiology lab was modified to include a substantial independent project, undertaken by groups of 2-4 students over a period of several weeks. Students were encouraged to think about community-based projects that involved interactions with local businesses, community agencies, etc. Sample descriptions of some of the projects
undertaken by students in the fall of 1999 are as follows:

a) One group performed a comparative water quality analysis, with special emphasis on coliform and fecal coliform bacterial counts, on several local water supplies (Connecticut River north of Northampton, Connecticut River south of Springfield, Lower Lake (a campus pond), South Hadley Sewage Treatment Plant effluent, and Puffer's Pond (a popular swimming hole). Lower Lake had the highest coliform counts, and perhaps surprisingly, there was no significant difference in the bacterial counts from the two different Connecticut River samples.

b) One group isolated and quantified coliphage viruses from raw sewage and treatment plant effluent. Many diverse viruses could be found in the raw sewage, but they were unable to isolate any from the effluent.

c) One group visited Cook's Dairy, a local farm in Hadley, and studied the succession of microorganisms over a period of six weeks from a sample of raw milk.

d) One group assayed the quantity and type of microorganisms found in various make-up products (mascara, lipstick, rouge, etc) that had been used and were sitting around in their purses, rooms, etc. Sponges or pads for applying make-up were found to retain particularly high numbers of bacteria.

e) One group purchased unwashed and "triple-washed" lettuce at the grocery store and sampled them for bacteria and fungi. There was no significant difference between the two samples - both were loaded with bacteria and fungi!

These group investigative projects proved to be quite successful, and they will be included in the laboratory when this course is offered again in the fall of 2000.

Biological Sciences 213 (Ecology and Evolution) and
Environmental Studies 200 (Environmental Science)
As requested in the proposal, we purchased a YSI 6820 field sonde with sensors for nitrate, ammonium, chlorophyll, pH, dissolved oxygen, temperature, and conductivity. The chlorophyll probe on the YSI was purchased in lieu of the Turner field fluorometer. We also purchased a Hansatech oxygen electrode.
This equipment has been used extensively in the Environmental Science course (ENVST 200), where water quality monitoring in Stony Brook has become a centerpiece lab. The ecology lab envisioned for Biology 213, for which this equipment was purchased, was developed instead for Environmental Science, a course that did not exist when the proposal was written. In addition, the equipment has been used for several independent and honors projects, including one by Laurel Moulton '01, who also received a grant-in-aid of research from the Society for Wetland Scientists for her work.
This equipment is also used heavily by students, staff, and faculty in the Center for Environmental Literacy (which also did not exist when the proposal was written). The data are integrated with GIS data on the campus and surrounding watershed to provide environmental analyses of the watershed. The pilot work supported by the ILI grant is being enhanced with new support for this project from the Mellon Foundation.
Finally, this equipment was used to complete the environmental impact assessment (EIA) of the Orchards Golf Course irrigation project as described in the proposal. This EIA involved 2 faculty members, 1 staff member, and 6 students, two of whom developed their work on this project into senior honors projects in the Geology Department.

Last Modified 27 September, 2000 Woodard Lab Home Page Craig T. Woodard