A new fluorescence reaction in DNA cytochemistry: microscopic and spectroscopic studies on the aromatic diamidino compound M&B 938.
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.
Stockert
J C
JC
Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Spain.
Trigoso
C I
CI
Cuéllar
T
T
Bella
J L
JL
Lisanti
J A
JA
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002875:Chromosomes / Q000737:chemistry / Q000648:ultrastructure; D002877:Chromosomes, Human / Q000737:chemistry / Q000648:ultrastructure; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006570:Heterochromatin / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D015394:Molecular Structure; D011067:Poly dA-dT; D051381:Rats; D013050:Spectrometry, Fluorescence; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000166:cytology
D005456:Fluorescent Dyes; D006570:Heterochromatin; C046869:4,4'-diamidinodiphenylamine; D011067:Poly dA-dT; D004247:DNA; D004159:Diphenylamine