Sebastian's Research: Past and Present
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Updated: May 5, 2002 I am currently working in Dr. Redfield's lab for my Masters thesis. We are trying to understand the mechanism of DNA uptake in Haemophilus influenzae. 'Natural competence' is the physiological state that enables cells to take up DNA from their environment. This process involves at least three steps: (1) DNA binding to the cell surface via some protein (complex?), (2) translocation of the DNA fragment across the cell membrane and into the cytoplasm, and (3) DNA degradation and/or recombination. This is a widespread process in bacteria. Some details of natural competence are known in certain species, but much remains to be determined. Haemophilus influenzae is a Gram-negative bacterium (i.e. it has two membranes and a thin cell wall) and was the first species to have had its genome completely sequenced. Haemophilus has a mechanism for taking up only conspecific DNA. This occurs via the recognition of a nine base pair 'uptake signal sequence' (USS) that is abundant throughout the genome. We don't know what genes are responsible for the first step in DNA uptake in Haemophilus (i.e. recognition of, and binding to, USSs on conspecific DNA). I have been using a variety of bioinformatic tools to characterize H. influenzae genes involved in natural competence, so that candidate genes can be targeted for mutational studies. Ideally, we would want some protein that localizes to the outer membrane and has a DNA binding motif. It could be that several proteins form a complex for USS recognition. Some of the genes regulated during competence development do have either signal peptides or transmembrane helices (I have yet to use a beta-barrel motif search program, which would be useful to identify proteins that localize to the outer membrane). My tentative plan is to knockout candidate genes and identify which protein(s) is responsible for specifically recognizing the USS. This will be useful in understanding the mechanism and evolution of natural competence in Haemophilus and perhaps in distantly related species as well. I have also done some DNA competition experiments in a comEA (TIGR accession number, HI1008) knockout that was constructed by the lab technician (HI1008 is homologous to a DNA binding protein, comEA, in Bacillus subtilis). When I actually have a working proposal, I will post it here. Before I started working on bacteria, I had a strong interest in plants. I did my Honours thesis in a plant genetics lab that worked on a self-incompatible tropical weed (Turnera sp.). At around the same time, I worked in a fungal genetics lab that did research on actin-binding proteins. Below, I have divided my previous areas of research into two sections. In the first section, I have provided some background info and the proposal for my Honours thesis. The second section contains information related to my work in the fungus lab, as well as some of the protocols I used.
Honours Thesis I did my Honours thesis with Dr. J. S. Shore at York University. Essentially I used a combination of genetics, molecular biology, and cell biology to study self-incompatibility in a homostylous mutant from an otherwise distylous species (genus: Turnera; family: Turneraceae). A "style" is a structure in a flower that contains stigmas which have receptors for pollen. Thus, a "distylous" species is defined as one that has two different floral morphs (different lengths of styles: short and long) in a population. The anthers in this type of species are also present in different lengths (i.e. short anthers in long-styled plants, and long anthers in short-styled plants). These different morphs are associated with a self-incompatibility response (i.e. shorts can cross with longs, and longs with shorts, but shorts cannot cross with shorts nor can longs cross with longs, to generate progeny). Pollen that is transferred to a style germinates. Germinated pollen grains form "pollen tubes", which grow down the style to fertilize the ovules. Fertilization, however, may be inhibited through the self-incompatibility response in order to promote outcrossing or to prevent inbreeding (for example, by inhibiting pollen tube growth).
Actin-Binding Proteins in Fungi My research in Dr. I. B. Heath's lab (York University) dealt with actin binding proteins (in particular, spectrin) in fungi. We were only at the preliminary stage of determining whether or not different fungal species even have spectrin or spectrin-like proteins. Below, I have provided some background information and a list of some protocols that I used in the lab. A large part of the background information presented here can be found in Alberts et al. (1994) Molecular Biology of the Cell, 3rd edition. I have not included any of the results I obtained, since there were a few problems with control experiments. Also, an important experiment I have tried is the "actin overlay", which is designed to detect those proteins that bind to actin filaments. Since I have had only limited success with this, I have not added the protocol (...but the principle is simple: after protein extraction and transfer to a membrane, wash the membrane with actin and use antibodies to detect binding). Background Info:
Protocols and Materials:
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