Influence of transcription and replication on the in situ resolution of immunoglobulin heavy-chain constant region genes: an interphase cytogenetics analysis.

An interphase cytogenetics analysis was performed to investigate 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 linear DNA distances was hybridized in situ and visualized by digital fluorescence microscopy. In interphase nuclei from phytohemagglutinin (PHA)-stimulated lymphocytes, the gamma genes were resolved as one to three signals per allele in the majority of nuclei, whereas in a minority, complex patterns of several signals per allele could be observed. The latter were restricted to nuclei in an early stage of the S phase, as assessed by hybridization experiments performed in cells grown in the presence of bromodeoxyuridine. To investigate whether the in situ resolution of the C gamma segments could vary as a function of the transcription activity of the locus, the C gamma probe was subsequently hybridized to nuclei from a mature B cell line (JVM-2), which produces gamma transcripts as shown by in situ RNA hybridization experiments. Primary human fibroblasts were further used as representative of a non-lymphoid cell type with transcriptionally inactive Ig genes. When Gl nuclei from the three cell types were compared in terms of the in situ resolution of the C gamma locus, JVM-2 cells were found to include the highest percentage of higher resolution patterns (three to five signals per allele in 28% of nuclei), fibroblasts the lowest (three signals per allele, 2%), while PHA-stimulated lymphocytes occupied an intermediate 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 gamma genes varies throughout the cell cycle and is influenced by the transcriptional activity of the locus. The variability of the resolution patterns observed appears to reflect different levels of chromatin packaging, 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 closely spaced, DNA segments.