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.

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.

Dr. Sossity Rapparell
Associate Professor of Immunology
Memorial Sloan-Kettering Cancer Center
1275 York Avenue, Box 40
New York, NY 10021
(212) 700-8900