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DNA labeled with non-radioactive markers have been used for hybridization with specific DNA or RNA either on filter or in cells and tissues. The presence of protruding markers on the probe DNA has been attributed to the cause for the loss of sensitivity and specificity of hybridization. In search of a non-protruding marker, we investigated the possibility of use of T-T dimer which can be generated easily in DNA and is a potent hapten as a marker for DNA. T-T dimer in DNA was generated by UV irradiation (254μm) for total of 4, 000-5, 000 joules/m2. For in situ hybridization; cells or tissues were first fixed either with Carnoy's fixative or formaldehyde and were usually treated with 0.2N HCl, then hybridized with the T-T dimerized DNA (T-T DNA). The hybridized T-T DNA was detected immunohistochemically using rabbit anti T-T DNA and peroxidase-labeled goat anti-rabbit IgG. With the Southern dot hybridization, the presence of 1 or less pg of complementary DNA can be detected. In cells and tissues, mRNA such as c-myc mRNA and growth hormone mRNA, and various viral DNA mRNA could be localized. The use of T-T as marker offers several advantages over other markers, it appears not interfere with the hybridization efficiency, simple to make and can be detected with high sensitivity.

Our preliminary immunohistochemical examination revealed that c-myc protein was localized in the cytoplasm of both normal and neoplastic cells. This finding did not correlate with the reported nuclear localization of c-myc protein by other analyses. To clarify this discrepancy, we investigated whether the cytoplasmic localization of c-myc protein in our preliminary immunohistochemical studies reflects true physiological habitat of the protein or the results of an artifact which occurred during tissue processing. For the immunohistochemical localization of c-myc protein, rabbit antisera against synthetic peptides which correspond to a portion of c-myc were utilized. Fixation conditions were examined by varying the temperature, the salt concentration, as well as the duration of fixation. With conventional fixation conditions used for the routine immunohistochemical localization of proteins, such as fixation with a solution of 4% formaldehyde with low salt concentration at low temperature, the c-myc protein was found in the cytoplasm of HL-60 cells, some gastric carcinoma cells, intestinal crypt epithelial cells and spermatogonia in seminiferous tubules from human and regenerating hepatocytes from rat. Whereas, when these cells and tissues were fixed at 42°C with a fixative containing 4% formaldehyde and a high concentration of NaCl, the c-myc protein was found in the nuclei of these cells. These results suggest that during fixation the c-myc protein translocates from nucleus to cytoplasm. To prove that these c-myc proteins were the product of the cells, in situ hybridization of c-myc mRNA was carried out using dinitrophenyl labeled c-myc cDNA. The gastric carcinoma cells which contained c-myc protein in their nuclei were morphologically similar to those contained the c-myc mRNA. The co-existance of c-myc protein and c-myc mRNA suggests that the c-myc protein found in the nuclei was the product of the cell.

To better describe the physiological state of cells, detection of specific mRNA by in situ hybridization at the cellular level is often demanded. In order to accomplish this reliably, various factors such as morphological preservation, retention of mRNA, accessibility of the labeled probe to the target mRNA and efficiency of the hybridization should be considered during the tissue processing. In this paper, using non-radioactive probes, we investigated the effects of protease treatment and the size of probe on the efficiency of in situ hybridization with mRNAs in frozen and paraffin-embedded tissue sections of the rat pituitary glands. As non-radioactive haptenic DNA probes, we used dinitrophenyl (DNP) -labeled pro-opiomelanocortin (POMC) DNA and thymine-thymine (T-T) dimerized prolactin cDNA. The signals were visualized by the indirect enzyme-immunohis-tochemistry. The best results were obtained when frozen sections of tissues fixed by 4% paraformaldehyde in phosphate buffered saline were mildly digested with protease and hybridized with the haptenized DNAs of about 200-400 base pairs.

PCNA/cyclin was originally described in proliferating mammalian cells as a nuclear protein with an apparent molecular weight of 33, 000-36, 000, and recently found to be a DNA polymerase-delta auxiliary protein. When the cDNA for PCNA/cyclin was cloned and analyzed, it was found that the protein consisted of 261 amino acids with a calculated molecular weight of 28, 700 and the amino acid sequence was well conserved during evolution both in animal and plant kingdoms. The highly homologous nature of PCNA/cyclin suggests that the protein plays an essential role in DNA replication in eukaryotes.