A new fluorescence reaction in DNA cytochemistry: microscopic and spectroscopic studies on the aromatic diamidino compound M&B 938.

Abstract

We describe the fluorescence properties and cytochemical applications of the aromatic diamidine M&B 938. Treatment of cell smears (chicken blood, Ehrlich ascites tumor, rat bone marrow, mouse mast cells, and Trypanosoma cruzi epimastigotes) with aqueous solutions of M&B 938 (0.5-1 microgram/ml at pH 6-7; UV excitation) induced bright bluish-white fluorescence in DNA-containing structures (interphase and mitotic chromatin, AT-rich kinetoplast DNA of T. cruzi), which was abolished by previous DNA extraction. DNA was the unique fluorescent polyanion after staining with M&B 938 at neutral or alkaline pH, other polyanions such as RNA and heparin showing no emission. M&B 938-stained mouse metaphase chromosomes revealed high fluorescence of the AT-rich centromeric heterochromatin, and strong emission of heterochromatin in human chromosomes 1, 9, 15, 16, and Y was found after distamycin A counterstaining. On agarose gel electrophoresis, M&B 938-stained DNA markers appeared as fluorescent bands. The 1.635-KBP fragment from DNA ladder revealed a higher emission value than that expected from linear regression analysis. Spectroscopic studies showed bathochromic and hyperchromic shifts in the absorption spectrum of M&B 938 complexed with DNA, as well as strong enhancement of fluorescence at 420 nm. In the presence of poly(dA)-poly(dT), the emission of M&B 938 was 4.25-fold higher than with DNA; no fluorescence was observed with poly(dG)-poly(dC). Experimental results and considerations of the chemical structure suggest that the minor groove of AT regions of DNA could be the specific binding site for M&B 938, which shows interesting properties and useful applications as a new DNA fluorochrome.

• Authors: J C Stockert, C I Trigoso, T Cuéllar, J L Bella, J A Lisanti
• Journal: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (J Histochem Cytochem) Vol. 45 Issue 1 Pg. 97-105 (Jan 1997) ISSN: 0022-1554 [Print] United States
• PMID: 9010474 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Fluorescent Dyes
• Heterochromatin
• 4,4'-diamidinodiphenylamine
• Poly dA-dT
• DNA
• Diphenylamine

Topics

• Animals
• Binding Sites
• Chickens
• Chromosomes (chemistry, ultrastructure)
• Chromosomes, Human (chemistry, ultrastructure)
• DNA (analysis, metabolism)
• Diphenylamine (analogs & derivatives, chemistry, metabolism)
• Electrophoresis, Agar Gel
• Fluorescent Dyes (chemistry, metabolism)
• Heterochromatin (chemistry)
• Humans
• Hydrogen-Ion Concentration
• Mice
• Microscopy, Fluorescence
• Models, Molecular
• Molecular Structure
• Poly dA-dT
• Rats
• Spectrometry, Fluorescence
• Trypanosoma cruzi (chemistry, cytology)