Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences.
The technique of nonradioactive in situ hybridization has been used to visualize the DNA and mRNA expression of human cytomegalovirus (HCMV) immediate early antigen (IEA) in a transfected rat fibroblast cell line. Expression of the transfected HCMV immediate early DNA can be induced by a cycloheximide treatment and is S-phase-dependent. In addition to cytoplasmic mRNA localization, a nuclear RNA hybridization signal was found. In a substantial part of the cells the nuclear signal was in the form of a "track," possibly showing transport of IEA mRNA from the site of transcription to the cytoplasm. The use of PCR-generated intron- and exon-specific probes in a double hybridization revealed that intron and exon mRNA sequences coexist in the nuclear RNA signal. This shows the applicability of multiple-color fluorescence hybridization to obtain information about the site of pre-mRNA splicing in the nucleus. In addition, by combining the technique of in situ hybridization with an immunocytochemical procedure we illustrate the possibility of visualizing transcribed mRNAs simultaneously with their translation products.
Raap
A K
AK
Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.
van de Rijke
F M
FM
Dirks
R W
RW
Sol
C J
CJ
Boom
R
R
van der Ploeg
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics*; D000939:Epitopes / Q000032:analysis; D005091:Exons; D005455:Fluorescent Antibody Technique; D016679:Genome, Viral; D006801:Humans; D017874:Immediate-Early Proteins; D007438:Introns; D008856:Microscopy, Fluorescence / Q000379:methods; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D014763:Viral Matrix Proteins; C044582:immediate-early proteins, cytomegalovirus; D003513:Cycloheximide