Differential gene expression in apoptosis: identification of ribosomal protein S29 as an apoptotic inducer.
To identify genes that are specifically involved in apoptosis, poly(A)(+) RNAs were isolated from untreated control rat thymocytes and from adriamycin-induced apoptotic thymocytes. Directionally cloned cDNA libraries were then constructed in UNIZAP-XR vectors followed by biotin-based subtractive hybridization. Three clones were confirmed to be differentially expressed by dot blotting. Sequence analysis revealed homology to two genes previously identified, whereas one clone was novel and did not have homology to any known sequence. One clone was identical to the ribosomal protein S29, and the other was homologous to L8 ribosomal protein. Northern blot analysis revealed a marked increase in the expression of mRNA encoding ribosomal protein S29 in the apoptotic thymocytes compared to the controls. Transfection studies revealed that enhanced S29 expression resulted in increased apoptosis in rat thymocytes and HeLa cells as assessed by various morphological and biochemical characteristics, including cell shrinkage, chromatin condensation, membrane blebbing, formation of apoptotic bodies, TUNEL, FACS, and internucleosomal DNA fragmentation. This was accompanied by upregulation of p53, Caspase 3, and bax, whereas bcl-2 was downregulated as revealed by Western blotting. The current findings provide the first hint of a role for ribosomal protein S29 in the apoptotic process.
Khanna
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Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India.
Reddy
V G
VG
Tuteja
N
N
Singh
N
N
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009707:Nucleosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D013950:Thymus Gland / Q000166:cytology; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein; D001616:beta-Galactosidase / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; C494826:BAX protein, human; C494828:Bax protein, rat; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D009707:Nucleosomes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012269:Ribosomal Proteins; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C079855:ribosomal protein S29; D004317:Doxorubicin; D001616:beta-Galactosidase; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases