当前位置： SCI文献检索 > CHROMOSOMA期刊下所有文献 > Preparation and imaging of nuclear spreads from cells of the zebrafish embryo. Evidence for large degradation intermediates in apoptosis.

Preparation and imaging of nuclear spreads from cells of the zebrafish embryo. Evidence for large degradation intermediates in apoptosis.

Abstract：

:We describe a method for preparing nuclear spreads from cells of live, unfixed zebrafish embryos at the late-gastrula (approximately 8000 cell) stage of development. The method consists of a sequence of four steps: (1) a slow, gentle lysis, in low to moderate salt concentration, of cells and then nuclei, to release DNA-containing fibres; (2) spreading of the released fibres by a transverse fluid flow; (3) electrostatic, and possibly also covalent, attachment of the spread fibers to poly(L-lysine)-coated glass microscope slides; and (4) continued incubation to produce periodic cleavage of the DNA within the fibres, apparently through activation of endogenous nucleases. The nuclear spreads are imaged with epifluorescence, at a spatial resolution approaching the Rayleigh limit (approximately 230 nm for blue light). The epifluorescent signal is provided from Hoechst 33,258 bound specifically to the DNA, from a dye-coupled antibody conjugate bound specifically to histone H1 in the fibres, or from a DNA nick end-labelling assay. The spontaneous cleavage of DNA-containing fibres in step (4) of the above procedure can be blocked by the chelating agents EGTA and EDTA, by the caspase-2,3,7 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde, and by the caspase-1,4,5 inhibitors N-acetyl-Tyr-Val-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone. These data suggest that the spontaneous cleavage of fibres is catalysed by nucleases that become activated through a caspase-mediated mechanism. The involvement of caspase-dependent nucleases would suggest that an apoptosis pathway is activated in the spreads during their prolonged incubation. If bona fide apoptosis is induced in living zebrafish embryos by treatment with camptothecin (a topoisomerase I poison), and then nuclear spreads are prepared, we observe a similar fragmentation of the spread fibres. However, in this case the fragmentation is more rapid and complete. We hypothesize that, during the early phase of apoptosis, one or more endogenous nucleases are activated by a caspase-mediated mechanism. The nuclease(s) then specifically recognize and cleave a susceptible, periodically repeating feature of interphase chromatin.

journal_name

Chromosoma

journal_title

Chromosoma

authors

Chan DW,Yager TD

doi

10.1007/s004120050280

subject

Has Abstract

pub_date

1998-03-01 00:00:00

pages

39-60

issue

eissn

0009-5915

issn

1432-0886

journal_volume

107

pub_type

杂志文章

在线工具

Preparation and imaging of nuclear spreads from cells of the zebrafish embryo. Evidence for large degradation intermediates in apoptosis.