Scientific Curriculum Vitae
John Doenan
345 Grebhorn Lane, Apt. 7C
Lookout, VA 98765
(617) 456-7889 (home)
(617) 543-2121
email: jdoenan@aol.com
Education
Ph.D, Graduation Date: June 31, 1997
Department of Immunology
Cornell University Medical College
Memorial Sloan-Kettering Cancer Center
1990
Bachelor of Science, Department of Biochemistry
University of Massachusetts, Amherst
Awards and Honors
1990-present
Research Fellowship from the Graduate School of
Cornell University Medical Center
1990
Magna Cum Laude
1987-1990
Dean's List
1986
John Danforth Leadership Award
Publications
John Doenan, Gustave Hall, and Sossity Rapparell. Coordinate Regulation of HLA Class II Genes: A Novel DNA Binding Complex. Molecular Immunology, Vol. 66, No. 11/12. pp. 490-511, 1998.
John Doe and Sossity Rapparell. Identification of Alternatively Spliced Transcripts for p50csk Which Are Diminished in Cell Lines from a Bare Lymphocyte Syndrome Patient. [In Preparation]
John Doenan, John Timpane, and Ned Thorpe. The Role of the Cytoskeleton and Intercellular Junctions in the Transcellular Membrane Protein Polarity of Bovine Aortic Endothelial Cells in Vitro. Journal of Cell Science (89): 88-99, 1991.
Abstracts
Sossity Rapparell, Nestor Gutierrez, John Doe, Lourdes Puente, Frances Ubarte, and Victor Cheung. Molecular Defects in HLA Class II Negative Congenital Immunodeficiency. Keystone Meeting on the Molecular biology of Human Genetic Disease. 1992: D 331
Research Experience
DNA: subcloning, minipreps, maxipreps, PCR, site-directed mutagenesis, footprinting, Sequencing, Gel Shifts, UV crosslinking, deletional analysis, library construction.
RNA: Northern, RNAse mapping, In vitro transcription, RT- PCR
Protein: SDS PAGE, Western Blots, Immunoprecipitation, Kinase assays, bacterial expression, ELISA.
Cell Culture: Transient transfections, CAT assays, retroviral infections, growth of transformed B cells, T cells, fibroblasts, monocytes, etc.
Library
Screening:
Lambda gt10, gt11, Lambda Dash, Lambda Zap express,
P1, pCDMB.
Current Research Project
My research has focused on the regulation of MHC class II
expression. In one set of experiments, I attempted to determine the
genetic defect in the MHC class II negative B cell line BLS-1,
derived from a Bare Lymphocyte Syndrome patient. Because the mutant
B cells were refractory to transfection, I could not rely on
complementation cloning. However, since the disease is autosomal
recessive, the unaffected parental line contains only one
functional allele at the disease locus. I developed a retroviral
insertion and inactivation strategy to randomly mutagenize the
parental line, converting it to MHC class II negative. Determining
where the retrovirus had integrated led me to the disease locus,
which for this complementation group was the tyrosine kinase CSK.
We analyzed the CSK locus in BLS-1 cells and did not find a
mutation in the CSK coding region. Similarly, no difference in RNA
expression in Northern blots and RNAse mapping experiments were
found, nor were there differences at the protein level as
determined through Western blotting, immunoprecipitations, and
kinase assays. However, I did discover novel start and stop sites
for CSK, and several alternatively-spliced clones. My current
hypothesis is that a rare alternatively-spliced CSK clone is
modulating the activity of MHC class II and its absence in BLS-1
cells is leading to the negative phenotype. I am currently testing
this in knockout mouse models.
In a separate set of experiments, I have characterized a nuclear
protein complex from B lymphoblastoid cell lines that binds to HLA
class II promoters as detected by electrophoretic gel mobility
shift assays (EMSA). This complex (C1) binds to at least three
independent sites in the proximal DRA promoter which have not been
identified previously as cis-acting elements. C1 is very abundant
in Burkitt's lymphoma cell lines, but less abundant in "normal" B
lymphoblastoid cell lines. The binding specificity of the C1
complex was analyzed using competition experiments and chemical
footprinting methods. Complexes with specificity similar to C1 also
bind the DPA and DQA promoters, suggesting that the C! complex is
involved in coordinate regulation of HLA class II genes. However,
mutation of the sequences in the DRA promoter that abolished
binding of the C1 complex had no effect on the ability of the DRA
fragment to drive transcription of the reporter gene in transient
expression or in vitro transcription assays. Nonetheless,
expression of C1 is reduced in cell lines with mutations affecting
class II transcription. In addition, complexes similar to C1 were
observed in nuclear extracts from all cell lines examined, but
minor differences in mobility appeared to correlate with class II
expression. Thus, the C1 complex may act as a positive trans-acting
factor in MHC class II expression.
References
Dr. Sossity Rapparell
Associate Professor of Immunology
Memorial Sloan-Kettering Cancer Center
1275 York Avenue, Box 40
New York, NY 10021
(212) 700-8900
Dr. Jason Fussell
Professor of Immunology
Memorial Sloan-Kettering Cancer Center
1275 York Avenue, Room K-18
New York, NY 10021
(212) 700-8900
Dr. Bengt Ivarsson
Professor of Immunology
Memorial Sloan-Kettering Cancer Center
1275 York Avenue, room K-56-X
New York, NY 10021
(212) 700-8900
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