1998 Summer HHMI-supported Participants-HHMI Undergraduate Program - Carnegie Mellon University

1998 Summer HHMI-supported Participants

Laura Conlin

Laura Conlin, Carnegie Mellon University
(Advisor: Dr. Elizabeth Jones)

Defining the functional domains of the PEP5 gene of Saccharomyces cerevisiae by in vitro mutagenesis

The PEP5 gene in Sacchromyces cerevisiae encodes a hydrophobic vacuolar peripheral membrane protein consisting of 1029 amino acids. The absence of Pep5p results in the absence of a discernible vacuole and disrupts the maturation of all vacuolar hydrolases. Pep5p is known to function at all three of the known pathways to the vacuole: between the Golgi and the endosome and also between the endosome and the vacuole in the secretory pathway, the non-endosomal pathway, and the cytoplasm to vacuole targeting (CVT) pathway. There are several motifs in Pep5p that are substantially similar other organisms including H. sapiens. Mutations in three of these motifs were created using in vitro methods to determine the functional domains of Pep5p. ATP binding site mutants were created using dut- ung- oligonucleotide-directed mutagenesis. PCR mutagenesis was used to create mutations in the Zinc finger motif as well as the area of homology to ESTs. The mutants will be tested to determine which motifs are important for function. Function will be examined by the APE test which shows whether the vacuole and secretory pathway are functional. Maturation of the pathway specific hydrolases: alkaline phosphatase (non-endosomal pathway) and aminopeptidase I (CVT pathway), will be examined by Western Blot in order to check if the pathways are functional and/or the vacuole is present.

Image of William Dirks

William Dirks, Western Washington Univ., Bellingham, WA
(Advisor: Dr. Robert F. Murphy)

Detection of Subpopulations in Images of Fluorescently Labeled Subcellular Structures via Cluster Analysis

When cellular images with fluorescently labeled substructures are collected it is not known whether the images come from one or more than one homogeneous population. If the images come from a number of subpopulations the systematic determination of these subgroupings could provide both insight into nature of the subcellular structure labeled and a method of determining the effects of substances on localization of the labeled structure. To determine the presence of subpopulations in fluorescence microscopy images, numerical features from the images were calculated transforming each image into a point in a feature space. These points were then placed into groupings using a maximum likelihood clustering method with the Akaike information criterion to determine the quality of the clustering (N. Ichimura, Robust Clustering Based on a Maximum-Likelihood Method for Estimating a Suitable Number of Clusters, Systems and Computers in Japan 28:10-23, 1997). The method was applied to images of Chinese Hamster Ovary cells labeled for Giantin, Lamp2, Tubulin, and DNA.

Elizabeth Litzinger

Elizabeth Litzinger, Carnegie Mellon University
(Advisor: Dr. Bruce Armitage)

Binding of Cyanine Dyes to Nucleic Acids

Binding of small organic molecules to double-helical DNA can be used for diagnostic and therapeutic applications. In the current work, optical spectroscopic techniques have been used to demonstrate that symmetrical cyanine dyes bind in the minor grove of DNA as face-to-face dimers. This binding mode is characterized by a preference for alternating A-T sequences and is most readily detected by the appearance of a blue-shifted absorbance band. Further evidence in support of a dimer-binding motif comes from the observation that two benzothiazole cyanines with different polymethine bridge lengths form mixed dimers ("heterodimers") in the presence of DNA. The heterodimer exhibits an absorption maximum at ca. 640 nm, intermediate between the values for the homodimers (592 nm and 679 nm for pentamethine- and heptamethine-bridged dyes, respectively).

Christopher Raymond

Christopher Raymond, Carnegie Mellon University
(Advisor: Dr. John Nagle)

The use of neutral density centrifugation and x-ray diffraction to investigate a possible change in molecular volume of DPPC under osmotic stress

We performed both neutral density centrifugation and x-ray diffraction techniques to investigate the gel phase of dipalmitoylphosphatidylcholine (DPPC). We then attempted to measure the number of water molecules per lipid and the molecular volume of the lipid for various osmotic pressures. By doing this we hoped to learn more about the structure and function of the lipid bilayers. The results of our experiments showed that these techniques produced errors that were too large to accurately describe how the number of water molecules per lipid changes with increasing osmotic stress. The results of the experiment also showed that the molecular volume of the lipid remains constant over a range of osmotic pressures. The value we measured for the molecular volume of the lipid was 1145 +/- 1.1 Angstroms.

Ankur Saxena

Ankur Saxena, University of Texas, Austin, TX
(Advisor: Dr. John Woolford)

Dominant Negative Alleles of the Yeast Nucleolar Protein Gene NOP4

Ribosome biogenesis in Saccharomyces cerevisiae involves a complicated series of events that includes processing of pre-rRNA and which leads to the creation of functional ribosomes. Among the many contributors to this process is Nop4p, an RNA recognition motif protein (RRM protein). Nop4p is involved in 60S ribosomal subunit biogenesis and is known to bind to RNA; beyond this, its exact function is unknown. To clarify this protein's role in ribosome biogenesis, a screen for dominant lethal mutations was initiated. Isolation of dominant lethal alleles of NOP4 followed by their characterization will facilitate the study of the protein's structure and function. Isolation of these alleles will also allow identification, through biochemical and genetic means, of molecules that interact with Nop4p. Through such detailed observation and analysis, it is hoped that the role of Nop4p in ribosomal biogenesis will be further elucidated.

Image of David Van Goor

David Van Goor, Carnegie Mellon University
(Advisor: Dr. William Brown)

Epitope Mapping of Toluene Diisocyanate--Human Serum Albumin Conjugates

Toluene diisocyanate (TDI) is a compound that is widely used in the commercial manufacture of polyurethane foams, elastomers, and coatings. TDI is a very reactive and volatile compound, causing it to be an occupational health concern. Exposure to TDI has been linked to a number of respiratory diseases, such as occupational asthma. Much current research has been focused upon understanding the biological processes involved in the immune response that follows exposure to TDI, particularly with respect to the production of immunoglobulins. Chemically modified proteins (such as human serum albumin, HSA) are thought to be involved in the triggering of an immunologic response. In our lab, a number of TDI/HSA conjugates have been developed that have been shown to have antigenic properties when exposed to the serum of workers who were diagnosed with TDI asthma. It is the goal of this project to determine how the modification of HSA by TDI leads to the formation of an antigen. Particularly, we hope to determine the individual sites where modification has occurred and (if possible) to isolate the smallest intact antigenic site (epitope) of the conjugate. The methods used were primarily enzymatic protein digestion, peptide separation using capillary electrophoresis and high-performance liquid chromatography, and the ELISA assay for antigenic activity.