PMID- 1689192
OWN - NLM
STAT- MEDLINE
DCOM- 19900320
LR  - 20210216
IS  - 0006-4971 (Print)
IS  - 0006-4971 (Linking)
VI  - 75
IP  - 4
DP  - 1990 Feb 15
TI  - Function of normal and mutated gamma-globin gene promoters in electroporated K562 
      erythroleukemia cells.
PG  - 990-9
AB  - We examined the importance of cis-acting regulatory elements of the human 
      gamma-globin gene promoter in a cell line (K562) where this gene normally 
      functions. A gamma-Globin promoter fragments were fused to the neomycin 
      phosphotransferase (neoR) gene in a plasmid-based vector and transiently 
      transfected by electroporation into K562 cells. Correctly initiated "A gamma-neo" 
      transcripts were detected with an S1 nuclease protection assay that was 
      internally controlled for transfection efficiency and RNA content. We first 
      optimized the conditions for electroporation, and then determined input DNA 
      concentrations that permitted study of gamma-promoter function in the linear 
      range of the assay. We discovered that a gamma-globin promoter fragment extending 
      from -299 to +36 (with respect to the transcription initiation site) was active 
      in this transient transfection assay, and that the expression of this promoter 
      was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to 
      position -199 did not significantly affect gamma-globin promoter function. 
      However, deletion to -160 reduced gamma promoter strength to 70% that of control, 
      deletion to position -130 to 19% that of control, and deletion to position -61 to 
      8.7% that of control. Three gamma-globin promoters containing mutations 
      associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 
      C----T, and -117 G----A) were not overexpressed in the K562 cell environment, 
      consistent with the hypothesis that these promoters are not overexpressed in 
      fetal erythroblasts, only adult erythroid cells. This system will allow us to 
      further dissect the roles of regulatory globin cis-acting DNA elements in fetal 
      erythroid cells.
FAU - Ulrich, M J
AU  - Ulrich MJ
AD  - Department of Medicine, Jewish Hospital at Washington University Medical Center, 
      St Louis, MO 63110.
FAU - Ley, T J
AU  - Ley TJ
LA  - eng
GR  - DK 38682/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Blood
JT  - Blood
JID - 7603509
RN  - 0 (Beta-Globulins)
RN  - 0 (gamma-Globulins)
RN  - 9007-49-2 (DNA)
RN  - 9034-63-3 (Fetal Hemoglobin)
SB  - IM
MH  - Beta-Globulins/genetics/metabolism/physiology
MH  - DNA/genetics
MH  - Electrophysiology
MH  - Fetal Hemoglobin/metabolism
MH  - Humans
MH  - Leukemia, Erythroblastic, Acute/genetics/*pathology/physiopathology
MH  - Leukemia, Experimental/genetics/*pathology/physiopathology
MH  - Leukemia, Myeloid/genetics/*pathology/physiopathology
MH  - Mutation
MH  - Promoter Regions, Genetic/*physiology
MH  - Transfection
MH  - gamma-Globulins/*genetics/metabolism/physiology
EDAT- 1990/02/15 00:00
MHDA- 1990/02/15 00:01
CRDT- 1990/02/15 00:00
PHST- 1990/02/15 00:00 [pubmed]
PHST- 1990/02/15 00:01 [medline]
PHST- 1990/02/15 00:00 [entrez]
AID - S0006-4971(20)85726-7 [pii]
PST - ppublish
SO  - Blood. 1990 Feb 15;75(4):990-9.