Poly A mRNA was isolated from five EBV B-CLL lines, H9, EPOS and ENEG cells and quantitated by uv and agarose gel electrophoresis. A northern blot using a Cu probe for the first exon of H chain gene was used to confirm that Ig specific mRNA was present. Poly A MRNA (5 ug) was used to make double stranded cDNA. The reaction for both first and second strand synthesis was carried out on thermal cycler. The cDNA was used as a target template while six oligonucleotide leader sequences and the Cu oligonucleotide were used in six PCR reactions to ascertain V region gene families. Presently the five cell lines all appear to belong to the VH3 family. Restriction analysis of the PCR products shows that at least 2 of 3 genes have a different sequence. Direct sequencing of the PCR product is planned but will require a modification in PCR conditions (asymetric). We also plan to design the necessary southern blot experiments to evaluate monoclonality in the third and fourth generation family members in B-CLL kindreds. We are also interested in developing in situ hybridizations with chromosome specific probes.
