Identification of the transcription termination site of the mouse nkx-1.2 gene: involvement of sequence-specific factors.
We have identified a transcription termination site in the 3' flanking region of the mouse nkx-1.2 gene. A downstream transcription regulatory element in the mouse nkx-1.2 gene was characterized by transferring its 3'-fragment into a chloramphenicol acetyl transferase (CAT) expression vector. Analysis of recombinant plasmids transfected into mouse NIH3T3 cells by CAT assay showed the possible region of regulation. There were two direct repeat structures containing poly(dG-dT) x poly(dC-dA) sequences (GT repeats) in this region. The precise location of transcription termination was mapped by nuclease S1 analysis of the transcripts from recombinant plasmids transfected into COSM6 cells. It was approximately 20 nucleotides upstream of the first GT repeat within the 5' sequences of the first element of the two direct repeats. Gel mobility shift assay and footprinting analysis demonstrated that nuclear DNA binding proteins bound specifically to the sequences where the termination occurred as well as the other sequences in the second element of the direct repeats. Southwestern analysis showed that 90-, 54-, 36- and 15-kDa nuclear proteins bound to the region of the termination. It is possible that one or more of those proteins are involved in blocking the elongation of the mouse nkx-1.2 gene transcript and then result in termination.
Hong
S B
SB
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Kim
S J
SJ
Noh
M J
MJ
Lee
Y M
YM
Kim
Y
Y
Yoo
O J
OJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C109095:Nkx1-2 protein, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases