Poly A mRNA was isolated from five EBV B-CLL linex, H9, EPOS and ENEG cells and quantitated by uv and agarose gel electrophoresis. A northern blot using a Vu probe for the first exxon 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 ass a target template while six oligonucleotide leader sequences and the Cu oligonucleotide were used in six PCR reactions to ascertain V region 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. The PCR products of the five EBV cell lines obtained with primers LS3 (leader sequence of the VH3 family) and C micro, both with an EcoRI restriction site added to the 5' end, were cloned and sequenced. Sequence reading was done with an IBI gel reader and sequence analysis using Macvector software in a Macintosh II computer. Analysis confirms that they belong to the VH3 family and also that there is a surprising homology between two cell lines. Sequencing of other colones of the same PCR products are under way in order to confirm these findings. We are still interested in developing a PCR quantitative analysis of the expression of VH genes in order to detect early disease in non-affected family members in kindred with familial B-CLL. Direct sequencing of the PCR product is planned but will require a modification in PCR conditions. We are also interested in developing in situ hybridizations with chromosome specific probes.
