Research Interests
My major research interests are in the area of eukaryotic gene regulation,
organization, and evolution. I have a strong interest in the genetic modifcation
and expression of altered genes by recombinant DNA techniques. Specific
research directions at the present time fall into three general areas:
DNA replication and integration of adeno-associated virus, structure-function
studies on carbonic anhydrase, and mitochondrial genetics.
Adeno-Associated Virus
Adeno-associated
virus (AAV) is a member of the Parvovirinae, small single-stranded
DNA viruses of vertebrates. AAV, a human virus, has the unique property
that a productive infection requires co-infection with a helper adenovirus
or herpes virus. In the absence of helper virus, AAV establishes a latent
infection in both cultured cells and in man via integration at a unique
site on human chromosome 19. AAV infection is apparently non-pathogenic.
Subsequent infection by a helper virus (or action by certain DNA-damaging
agents) leads to rescue of the integrated AAV genome, AAV DNA replication
and virion production. This ability to establish a latent infection without
pathogenesis gives AAV significant potential as a gene therapy vector.
My laboratory is collaborating with Dr. Kenneth I. Berns on studies using
a cell-free system to define AAV's requirements for cellular proteins during
in vitro AAV DNA replication. Net synthesis of AAV DNA occurs in
this cell-free system. We are particularly interested in the requirements
for DNA binding proteins and proteins involved in DNA repair functions.
In addition, a plasmid-based model of chromosomal integration has been
developed; the results indicate cell-free extracts can support low
levels of integration into a plasmid target. We will determine the constituents
of the cell-free extract necessary for this integration event. Finally,
we have a general interest in the development of AAV-based vectors for
use in gene therapy, and several projects are in initial stages.
Carbonic Anhydrase
In
a second area, we have cloned several of the human and mouse carbonic anhydrase
genes and carried out in vitro mutagenesis of the various proteins.
Further work, done in collaboration with Dr. David Silverman, determined
the effect of specific amino acid replacements on enzymatic activity, and
has allowed us to undertake an extensive analysis of structure-function
relationships in the carbonic anhydrases. Our groups are interested in
the role played by the various carbonic anhydrase isozymes in different
tissues and are examining tissue-specific expression of carbonic anhydrase
genes. Experiments leading to knockout and replacement of the endogenous
carbonic anhydrases with modified forms via transgenic techniques and development
of isozyme specific inhibitors are in progress. We are initiating
studies on phenylalanine hydroxylase, another metaloenzyme where the mass
spectrometer methods developed for carbonic anhydrase can be usefully applied.
This enzyme plays an essential role in brain development.
Mitochondrial Inheritance
Finally, my laboratory has a
long-standing interest in mitochondrial DNA (mtDNA) inheritance. We have
examined mitochondrial segregation and amplification during oogenesis and
after microinjection of genetically marked mitochondria into mouse oocytes.
We have successfully constructed heteroplasmic mice containing two different
types of mitochondria. Female heteroplasmic mice transmit both mtDNA genotypes
to their offspring. The ratio of the two mtDNAs varies in different offspring,
suggesting an early segregation event in development affects mtDNA distribution
in the embryo. In addition, there is a clear tissue bias in distribution
of the two mtDNA genotypes within individual animals. These experiments
are being extended to develop a technique for introducing in vitro
modified mtDNA into the female germ line, with the ultimate goal of producing
animal models for human mitochondrial diseases. We have also developed
methods for detecting mtDNA deletions in animals, to understand both how
the mutations arise and to create cell culture systems which could yield
deleted mtDNA molecules that could then be reintroduced into mouse embryos.
