Student Research Projects

Insertion of the Escherichia Coli Phosphofructokinase A (PFKA) Gene into a plasmid, pRSETA

Brian Fiske ’03

We attempted to insert the Escherichia Coli (E. Coli) gene for Phosphofructokinase A (PFKA), an enzyme involved in glycolysis, into the plasmid pRSETA, place it under the control of the easily-inducible T7 RNA Polymerase, and his-tag the gene. We first designed appropriate primers and used PCR to amplify the PFKA gene with added restriction sites (HindIII and BamH1) at either end from several different DH5D K12 chromosomal DNA samples, some of which we had prepared ourselves. We then cut the gene, along with a sample of pRSETA, with the restriction enzymes HindIII and BamH1. Then we “gel purified” the resulting DNA, a mix of unwanted smaller fragments as well as the properly designed cut plasmid and gene. We then ligated the gene and the plasmid together, and chemically transformed the resulting ligation into DH5D competent cells. After screening numerous colonies for the presence of plasmid DNA using a miniprep and a restriction analysis using HindIII and BamH1, we had hoped to find evidence of at least one successful transformation of the pRSETA plasmid with the PFKA insert. However we have come up with no evidence of a successful clone, and after redoing the procedure many times as well as eliminating many sources of error, we have decided to abandon the project with the conclusion that the cloning of this gene is very difficult or impossible to carry out by our limited methods.

The Influence of DNA Mismatch Repair on the Types of p53 Mutations Found in Msh2 Null and Msh2/Atm Double Null Transformants

Genevieve Chow ’03

Bone marrow that was harvested from Msh2 null and Msh2/Atm double null mice were previously infected by the oncogenic Abelson murine leukemia virus and grown until they became fully established cell lines in the Rosenberg Lab. By looking into exon 11 of the tumor suppressor gene, p53, I sought to determine if the absence of DNA Mismatch Repair (MMR) influences the types of p53 mutations found in Msh2 null and Msh2/Atm double null transformants. In this lab, I first used Polymerase Chain Reaction (PCR) to amplify samples isolated from 8 different cell lines. To be certain that the region had been amplified, I ran the PCR products on agarose gel and purified the samples. I then prepared two vectors: vBluescript SK+ and dTTP, which were cut with EcoRV. Next, I ligated the vector and PCR product, first with T4 DNA ligase and then Quick Ligase. In this study, the PCR cycling for all cell lines succeeded. Unfortunately, the T4 DNA ligase that I used was defective, thus my transformations failed to produce any colonies. I then switched to using Quick Ligase; however, due to the limited time in the fall, I was only able to do produce colonies for 18-63, 18-64, 22-60, and 22-90. In winter term, I then proceeded to grow the colonies in overnight cultures, isolate the DNA, and finally, do restriction analyses on them (first by using EcoRI and HindIII, then PVUII). Since I was unable to yield any inserts, I did new transformations for all cell lines (except 18-65); however, no colonies grew. I have decided not to continue working on the project for the remaining school year.

Paramecium bursaria chlorella Virus 1 and Bioinformatics: Attempts to Discover Patterns and Evolutionary Trends from Viral Protein Sequences.

Bryce Kaufman ’03

This bioinformatics research project sought to discover trends, patterns, or an understanding of the evolutionary history of the Paramecium bursaria chlorella virus 1 by using individual viral protein sequences and close protein homologues. This data was downloaded, aligned by a multiple-alignment program, Clustal-W, and then the Clustal-W data output was used to make evolutionary trees. Unfortunately, I was not able to find any clear patterns or to derive an understanding of the complete viral genome history, and only able to make individual protein analysis based on the trees.

Assessing Differential Gene Expression of Drosophila Melanogaster Using Differential Display Technique

Joana Damásio ’03

This research project was intended to analyze the differences in gene expression at different life stages of drosophila melanogaster by using a differential display approach. I focused on the adult and pupae stages. The gene expression of the larvae and egg stages were not analyzed because the larvae and eggs were too small to collect. First I isolated RNA from two different generations of adult flies (separately), young adults (recently enclosed adults), and pupae. Then I synthesized cDNA by reverse transcription in the PCR. The cDNA was then amplified in a PCR reaction. Next, I ran the amplified cDNA in a denaturing polyacrylamide gel. The gel did not come out as well as expected: few bands were visible. The bands that were visible, those of the pupae sample, came out as expected. The bands of both stocks of adults and the young adults did not come out. This is probably because not enough cDNA was present. Next term I plan to isolate RNA from more flies (maybe six instead of two) in order for a larger quantity of RNA to be extracted. Then more cDNA would be synthesized and amplified. I will also change the MgCl2 and dNTP concentrations of the PCR amplification reaction in order to achieve optimal results. If bands are shown, I will cut the bands of interest out of the gel, reamplify them and sequence them. Taking in consideration the technical difficulties of the procedures involved in this project, I think overall it was successful.

Purifying, Testing and Mutating VH1

Marianna Kleyman ’03

After properly isolating, purifying, and testing the activity of a phosphatase (VH1) from Vaccinia, I began making point mutations in its DNA in order to ascertain how its structure affects its function. VH1 is a gene from a virus called Vaccinia, which is a homologue of Smallpox, but harmless to humans. I was given the H1 gene inserted into a vector (pET 16b) and I transformed it into E. Coli bacteria in order to express protein. This gene codes for a dual-specificity phosphatase (it acts on both tyrosine and serine-threonine), which has a homologue in humans (VHR). My goal is to make point mutations around the active site of VH1 and see how they affect its function. In order to determine which amino acids to mutate, I looked at the active site of VHR (whose structure is known) and compared these amino acids to those in VH1 – I ran a BLAST search to compare the codes of the two homologues. The first mutations I decided on are g113r, g113a, r116s, and r116k. I succeeded in inserting the gene into E. Coli cells as shown by the Restriction Digest, and the protein is being over-expressed as shown by an SDS PAGE gel analysis. After many trials, I purified VH1 protein and found out its concentration by using a standard curve for nitrophenol phosphate. I attempted to mutate the original DNA using a PCR reaction and specially-designed primers, but I have not been able to get enough mutant DNA to use in transforming cells.

Determining the Genotoxicity of Certain Chemicals Through the Somatic Mutation and Recombination Test in Drosophila melanogaster

Sharon Lawrence ’03

Through the w / w+ somatic mutation and recombination test (SMART), I hope to determine the genotoxicity — the ability to produce mutations — of certain chemicals that are suspected to be carcinogenic. The SMART determines genotoxicity by exposing larvae to different concentrations of the chemicals being tested. The assay takes advantage of the opportunity to expose the imaginal discs of the larvae to the chemical, and in doing so, hopefully mutate at least one cell. The imaginal discs are groups of cells that reproduce through mitosis and eventually differentiate into the different parts of the fly’s body. If even one cell is successfully mutated, its daughter cells will carry the same mutation, eventually growing into a region of mutated cells among normal cells. The chemicals that I hope to test are potassium chromate (K2CrO4), a chemical proven to be genotoxic through the wing-spot test; juglone (5-hydroxy-2 methyl- 1,4 naphtoquinone), a natural chemical proven to be genotoxic in the eye-spot test; acetaldehyde, a suspected mutagen with many uses, from manufacturing disinfectants and explosives, to flavoring foods such as desserts and soft drinks; and ethidium bromide, a dye used in gel electrophoresis that is also an expected mutagen. Last term, I decided to test these chemicals using the 48-hour feeding method. This method requires separating three-day old larvae from the media they are in and putting them in vials containing chemical-treated media. This proved to be very difficult, however, so I tried the chronic feeding method this term. For this method, I allowed the flies to mate for a few days in vials containing untreated media, then transferred them to vials containing chemical treated media. Once the new flies emerge, I examined their eyes for the presence of white spots, which should occur if the chemicals are mutagenic. I performed the assay three times, but each time I failed to obtain any results. Next term, however, I plan on continuing my attempts to test potassium chromate, and hopefully also the other chemicals.

Using Bioinformatics and PERL Programming to Categorie and Simplify the Process of QTL Research for Specific Diseases

Ayodele Adesanya ‘05
The Jackson Laboratory Summer Student Program

from www.jax.org/education/ss04/adesanya.html

The PositionaL Candidate Analysis Display (PLAD) is a database of quantitative trait loci (QTL) available online that is used by researchers to identify disease-related genes. QTL are chromosome areas that contain genes involved in the expression of a trait, in this case various diseases. My main objective for my project was to redesign PLAD to make it more user friendly. These modifications included reorganizing the system, modifying the query options and using a color-coding scheme to present the data more clearly. Changes made to the query options will allow researchers to ask for only those genes that have specific characteristics or attributes. The color-coding will make it easier for researchers to differentiate between genetic sequences that are in coding areas of a gene (exons) and those in non-coding areas (introns). I updated the database by adding new disease QTL, focusing on those QTL related to spinal orthopedic conditions like scoliosis and osteoporosis. All of these modifications will decrease the time needed to narrow down thousands of genes to specific genes of interest that can be tested. Hopefully this will lead to more time for researchers to spend on actual gene investigations and, eventually, move discoveries.