INFLUENCE OF TRANSCRIPTION AND REPLICATION ON THE IN-SITU RESOLUTION OF IMMUNOGLOBULIN HEAVY-CHAIN CONSTANT-REGION GENES - AN INTERPHASE CYTOGENETICS ANALYSIS

in turn are likely to influence the probe accessibility to its target. These observations are relevant for the interpretation of data from i nterphase cytogenetics analysis of independent, but closely spaced, DN A segments. An interphase cytogenetics analysis was per formed to inve stigate whether replication and transcription could influence in situ resolution of immunoglobulin (Ig) heavy chain constant region genes. A plasmid probe recognizing five C gamma segments separated by known li near DNA distances was hybridized in situ and visualized by digital fl uorescence microscopy. In interphase nuclei from phytohemagglutinin (P HA)-stimulated lymphocytes, the gamma genes were resolved as one to th ree signals per allele in the majority of nuclei, whereas in a minorit y, complex patterns of several signals per allele could be observed. T he latter were restricted to nuclei in an early stage of the S phase, as assessed by hybridization experiments performed in cells grown in t he presence of bromodeoxyuridine. To investigate whether the in situ r esolution of the C gamma segments could vary as a function of the tran scription activity of the locus, the C gamma probe was subsequently hy bridized to nuclei from a mature B cell line (JVM-2), which produces g amma transcripts as shown by in situ RNA hybridization experiments. Pr imary human fibroblasts were further used as representative of a non-l ymphoid cell type with transcriptionally inactive Ig genes. When G1 nu clei from the three cell types were compared in terms of the in situ r esolution of the C gamma locus, JVM-2 cells were found to include the highest percentage of higher resolution patterns (three to five signal s per allele in 28% of nuclei), fibroblasts the lowest (three signals per allele, 2%), while PHA-stimulated lymphocytes occupied an intermed iate position between the other two cell types (three or four signals per allele, 15%). The data show that the in situ resolution of Ig C ga mma genes varies throughout the cell cycle and is influenced by the tr anscriptional activity of the locus. The variability of the resolution patterns observed appears to reflect different levels of chromatin pa ckaging, which in turn are likely to influence the probe accessibility to its target. These observations are relevant for the interpretation of data from interphase cytogenetics analysis of independent, but clo sely spaced, DNA segments.