Coexpression of gamma and beta globin mRNA in cells containing a single human beta globin locus: results from studies using single-cell reverse transcription polymerase chain reaction.
We developed a method detecting globin gene expression in single cells using reverse transcription polymerase chain reaction. epsilon and gamma globin cDNAs are coamplified by an epsilon gamma primer set whereas gamma and beta globin cDNAs are coamplified by a gamma beta primer set and the individual globin cDNAs are distinguished by restriction enzyme digestion. Analysis of RNA preparations from human fetal liver, neonatal red blood cells (RBCs), or adult RBCs showed the expected mRNA species for each stage of human development. Analysis of single cells from a human erythroleukemia line coexpressing gamma and beta globin chains showed heterogeneity in gamma and beta mRNA contents. The method was subsequently used to test whether only one or more than one globin genes are expressed in cells that contain a single human beta globin locus. We found that about 50% of single cells from MEL x fetal erythroid cell hybrids containing a single human beta globin locus coexpressed gamma and beta globin mRNA. This finding is best explained by assuming that both gamma and beta genes are simultaneously transcribed from the same beta globin locus implying that the LCR can simultaneously interact with more than one globin gene promoter.
Furukawa
T
T
Department of Medicine, University of Washington, Seattle 98195.
Zitnik
G
G
Leppig
K
K
Papayannopoulou
T
T
Stamatoyannopoulos
G
G
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D005796:Genes; D005914:Globins / Q000235:genetics*; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D013045:Species Specificity; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger; D005914:Globins