Characterization of apoptosis-like endonuclease activity in avian thymocytes

Khaled Machaca, Mark M. Compton

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibito aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.

Original languageEnglish
Pages (from-to)15-22
Number of pages8
JournalBiology of the Cell
Volume76
Issue number1
DOIs
Publication statusPublished - 1992
Externally publishedYes

Fingerprint

Endonucleases
Thymocytes
Apoptosis
Chickens
Agar Gel Electrophoresis
Erythrocytes
DNA
Nuclear Proteins
Cell Nucleus
Flow Cytometry
Aurintricarboxylic Acid
DNA Cleavage
Edetic Acid
Glucocorticoids
Chromatin
Zinc
Fluorescence
Ions
Acids

Keywords

  • apoptosis
  • endonuclease
  • flow cytometry
  • thymocytes

ASJC Scopus subject areas

  • Cell Biology

Cite this

Characterization of apoptosis-like endonuclease activity in avian thymocytes. / Machaca, Khaled; Compton, Mark M.

In: Biology of the Cell, Vol. 76, No. 1, 1992, p. 15-22.

Research output: Contribution to journalArticle

@article{1e46bcb57d9949b0ae28e02d50f78b7b,
title = "Characterization of apoptosis-like endonuclease activity in avian thymocytes",
abstract = "In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibito aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.",
keywords = "apoptosis, endonuclease, flow cytometry, thymocytes",
author = "Khaled Machaca and Compton, {Mark M.}",
year = "1992",
doi = "10.1016/0248-4900(92)90190-C",
language = "English",
volume = "76",
pages = "15--22",
journal = "Biology of the Cell",
issn = "0248-4900",
publisher = "Portland Press Ltd.",
number = "1",

}

TY - JOUR

T1 - Characterization of apoptosis-like endonuclease activity in avian thymocytes

AU - Machaca, Khaled

AU - Compton, Mark M.

PY - 1992

Y1 - 1992

N2 - In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibito aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.

AB - In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibito aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.

KW - apoptosis

KW - endonuclease

KW - flow cytometry

KW - thymocytes

UR - http://www.scopus.com/inward/record.url?scp=0027052613&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027052613&partnerID=8YFLogxK

U2 - 10.1016/0248-4900(92)90190-C

DO - 10.1016/0248-4900(92)90190-C

M3 - Article

C2 - 1338188

AN - SCOPUS:0027052613

VL - 76

SP - 15

EP - 22

JO - Biology of the Cell

JF - Biology of the Cell

SN - 0248-4900

IS - 1

ER -