Expression from herpesvirus promoters does not relieve the intron requirement for cytoplasmic accumulation of human beta-globin mRNA.
Expression plasmids were constructed in which the human beta-globin gene or a variant of it precisely lacking its two introns was transcribed from its own promoter, the herpes simplex virus type 1 thymidine kinase (HSV-tk) promoter, or the immediate early promoter of human cytomegalovirus (CMV-IE). Forty two hours after transfection of these plasmids into monkey kidney cells, nuclear and cytoplasmic RNA were isolated. Quantitative S1 nuclease mapping and primer extension analysis were used to determine the relative abundances, cellular locations, and leader sizes of the accumulated beta-globin RNAs. Whereas transcripts of all of the intron-containing genes accumulated in the cytoplasm to high levels, transcripts of their cDNA variants were neither stably maintained in the nucleus nor accumulated in the cytoplasm, irrespective of the promoter from which transcription was driven. We conclude that the intron requirement for cytoplasmic accumulation of beta-globin RNA can not be circumvented by synthesis from either the promoter of the intronless HSV-tk gene or the CMV-IE promoter.
Yu
X M
XM
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Gelembiuk
G W
GW
Wang
C Y
CY
Ryu
W S
WS
Mertz
J E
JE
D000956:Antigens, Viral / Q000235:genetics; D015139:Blotting, Southern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D005914:Globins / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins; D007438:Introns / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics
D000956:Antigens, Viral; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; C044582:immediate-early proteins, cytomegalovirus; D005914:Globins; D013937:Thymidine Kinase