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Gregory A. Buck (Principal Investigator for Institute)
BBSI project: How can sequenced
genomes from pathogenic microbes be used to develop vaccines?
The recent availability of sequenced
genomes from pathogenic microbes offers an entirely new way to develop
vaccines. Bioinformatic tools can be used to analyze the genomes
of pathogens for the location and structures of encoded proteins.
Many surface proteins identified in this way are candidates for
vaccines. All those candidate protein genes can be PCR amplified
and expressed in E coli. The purified proteins will be tested for
raising immune responses. This from silico to "wet" laboratory approach,
termed reverse vaccinology, facilitates the development of vaccines
and reduces the need to cultivate pathogens, as required by conventional
methods of vaccine discovery.
Other research interests (see web
page for more details)
Unusual mechanisms of gene expression and RNA maturation
in the simple eukaryotes Trypanosoma cruzi and Pneumocystis carinii
Research in this lab focuses on unusual mechanisms of gene expression
and RNA maturation in simple eukaryotes. Two experimental models,
Trypanosoma cruzi and Pneumocystis carinii, are currently being
examined. T. cruzi is the causative agent of Chagas' Disease in
Latin America where approximately 25 million people are at risk
for developing the disease. It is a member of the protozoan order
Kinetoplastida which diverged early in evolutionary history from
other eukaryotes, and as a result exhibits extremely unusual mechanisms
of gene expression and RNA maturation. An understanding of these
unusual strategies for gene expression provides a snapshot of the
primitive processes from which homologous mechanisms in higher eukaryotes
evolved. Specifically, we are studying the mechanisms of transcription
promotion and the process of MRNA maturation in T cruzi. We have
found that T cruzi transcription promoters are unusual, lacking
even the canonical TATA box. We have recently developed a genetic
system for the expression of erogenous genes in T cruzi, and have
begun to use this system to dissect cloned T cruzi promoters by
site directed mutagenesis and DNA transfection. We have also shown
that RNA maturation in T. cruzi is also unusual in that all nuclear
mRNAs are chimeric; i.e., they are the products of a bi-molecular
or trans-splicing event. Trans-splicing resembles MRNA splicing
of higher eukaryotes but differs in its bi-molecular nature. We
are using our genetic system to introduce specifically mutated genes
into T cruzi to dissect this process genetically. We have also developed
and applied T. cruzi nuclear extracts that perform some of the MRNA
maturation functions to identify particles and protein factors that
participate in this novel process. Again, an understanding of these
unusual processes provides insight into the mechanisms of RNA metabolism
of higher eukaryotes.
P. carinii is the most important opportunistic pathogen of AIDS
patients. This organism is poorly understood due to the relatively
recent advent of AIDS and because it remains impossible to culture.
We have recently shown, by pulsed field gel electrophoresis, that
the genome of P. carinii is small for a eukaryote, and contains
only 12-15 small chromosomes. We are currently constructing a complete
library of P. carinii chromosomes in Yeast Artificial Chromosome
vectors. Moreover, direct sequence analysis of the P. carinii ribosomal
RNAs indicates that this organism is best classified as a fungus.
During these studies, we found that each of the P. carinii ribosomal
genes bears an unusual intron which, when transcribed in vitro from
clones, self-splices in the absence of proteins. This and additional
work confirmed that this intron is a member of the unusual class
of group-I introns. Since no group-I introns have been found in
higher eukaryotes, we are exploring the possibility that specific
inhibitors of this self-splicing reaction are potential chemotherapeutic
agents.
Nucleic acid biochemistry
Finally, we are working actively in the field of nucleic acid biochemistry.
The lab includes the VCU Health System and VCU Nucleic Acids Core
Facility, which maintains and operates several automated DNA/RNA
synthesizers and sequencers and develops methodology to enhance
this technology. Specifically, we are optimizing protocols for synthesis
and sequencing of nucleic acids and are working on methodology for
application of antisense RNA and ribozymes to artificially downregulate
genes in vivo.
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