EVALUATION OF THE SENSITIVITY OF THE TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE-IMMUNOGOLD TECHNIQUE ON BALBANI RING GENES

Recently, we developed the terminal deoxynucleotidyl transferase (TdT) -immunogold technique for in situ detection of DNA molecules. In this study the potential value and the limitations of the method were evalu ated using the giant polytene chromosomes from Chironomus tentans sali vary glands. Emphasis was put on the Balbiani rings (BRs), specialized chromosomal sites with exceptionally intense synthesis of large mRNA molecules. Immunolabeling was recorded not only over the bands and int erbands of the polytene chromosomes but also over the BR structures, I n the BRs, gold particles were present over segments of active transcr iption units, each with a central chromatin axis and a number of growi ng RNP products attached to the axis. One third of the transversely se ctioned transcription units showed labeling in the central parts, i.e. , where the unfolded chromatin axis is located, whereas the growing RN P fibers remained unlabeled. The absence of labeling of the RNP fibers is not likely to be due to lack of accessibility, because anti-RNA an tibodies readily decorated the RNP fibers. The nuclear sap and cytopla sm displayed no significant label. These results clearly indicate that the TdT-immunogold technique is specific for DNA and detects not only DNA in compacted chromatin but also fully extended DNA. Its ability t o efficiently label a single DNA molecule demonstrates the method's ve ry high sensitivity.